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Paper by Ramon K Tabtiang Brent O Cezairliyan Robert A Grant Jesse C Cochrane and Robert T Sauer - Department of Biology Massachusetts Institute of Technology Cambridge MA 02139
Presented by Gayathri Priya Ravichandran
Consolidating critical binding
determinants
by noncyclic rearrangement of protein
secondary structure
Structure
bull Lot of basicsbull Objectivebull Introductionbull Methodsbull Some discussionsbull Referencebull Questions
Basics ndash Levels of protein structurebull Secondary Structure The secondary structure of a
protein describes certain repetitive local conformations that are found in most peptide chains The secondary structure does not describe the actual folding the protein in three dimensions but instead illustrates the structure of small regions of the peptide
bull Primary Structure The primary structure of a biological molecule is the exact specification of its atomic composition and the chemical bonds connecting those atoms
Levels of protein structure
1 Primary ndash amino acid sequence and disulfide bonds2 Secondary ndash Local folding to form regular elements3 Tertiary ndash 3 Dimensional folded structure4 Quaternary ndash Tertiary structure of multimeric proteins
Alpha Helix
bull It is a secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( hydrogen bonding)
Beta Sheet
bull The β sheet (also β-pleated sheet) is the second form of regular secondary structure in proteins bull Beta sheets consist of beta strands connected
laterally by at least two or three backbone hydrogen bonds forming a generally twisted pleated sheetbull A beta strand (also β strand) is a stretch of
polypeptide chain typically 3 to 10 amino acids long with backbone in an extended conformation
Beta sheet
Operatorbull In genetics an operator is a segment of DNA to which a
transcription factor binds to regulate gene expression The transcription factor is typically a repressor which can bind to the operator to prevent transcription
bull Transcription is the first step of gene expression in which a particular segment of DNA is copied into RNA (mRNA tRNA or rRNA) by the enzyme RNA polymerase
Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the
expression of one or more genes by binding to the operator or associated silencers
Transcription
Arc molecule
bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally
defined by their ability to be transcribed in the presence of protein synthesis inhibitors
Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are
short chains of amino acid monomers linked by peptide (amide) bonds
bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Structure
bull Lot of basicsbull Objectivebull Introductionbull Methodsbull Some discussionsbull Referencebull Questions
Basics ndash Levels of protein structurebull Secondary Structure The secondary structure of a
protein describes certain repetitive local conformations that are found in most peptide chains The secondary structure does not describe the actual folding the protein in three dimensions but instead illustrates the structure of small regions of the peptide
bull Primary Structure The primary structure of a biological molecule is the exact specification of its atomic composition and the chemical bonds connecting those atoms
Levels of protein structure
1 Primary ndash amino acid sequence and disulfide bonds2 Secondary ndash Local folding to form regular elements3 Tertiary ndash 3 Dimensional folded structure4 Quaternary ndash Tertiary structure of multimeric proteins
Alpha Helix
bull It is a secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( hydrogen bonding)
Beta Sheet
bull The β sheet (also β-pleated sheet) is the second form of regular secondary structure in proteins bull Beta sheets consist of beta strands connected
laterally by at least two or three backbone hydrogen bonds forming a generally twisted pleated sheetbull A beta strand (also β strand) is a stretch of
polypeptide chain typically 3 to 10 amino acids long with backbone in an extended conformation
Beta sheet
Operatorbull In genetics an operator is a segment of DNA to which a
transcription factor binds to regulate gene expression The transcription factor is typically a repressor which can bind to the operator to prevent transcription
bull Transcription is the first step of gene expression in which a particular segment of DNA is copied into RNA (mRNA tRNA or rRNA) by the enzyme RNA polymerase
Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the
expression of one or more genes by binding to the operator or associated silencers
Transcription
Arc molecule
bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally
defined by their ability to be transcribed in the presence of protein synthesis inhibitors
Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are
short chains of amino acid monomers linked by peptide (amide) bonds
bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Basics ndash Levels of protein structurebull Secondary Structure The secondary structure of a
protein describes certain repetitive local conformations that are found in most peptide chains The secondary structure does not describe the actual folding the protein in three dimensions but instead illustrates the structure of small regions of the peptide
bull Primary Structure The primary structure of a biological molecule is the exact specification of its atomic composition and the chemical bonds connecting those atoms
Levels of protein structure
1 Primary ndash amino acid sequence and disulfide bonds2 Secondary ndash Local folding to form regular elements3 Tertiary ndash 3 Dimensional folded structure4 Quaternary ndash Tertiary structure of multimeric proteins
Alpha Helix
bull It is a secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( hydrogen bonding)
Beta Sheet
bull The β sheet (also β-pleated sheet) is the second form of regular secondary structure in proteins bull Beta sheets consist of beta strands connected
laterally by at least two or three backbone hydrogen bonds forming a generally twisted pleated sheetbull A beta strand (also β strand) is a stretch of
polypeptide chain typically 3 to 10 amino acids long with backbone in an extended conformation
Beta sheet
Operatorbull In genetics an operator is a segment of DNA to which a
transcription factor binds to regulate gene expression The transcription factor is typically a repressor which can bind to the operator to prevent transcription
bull Transcription is the first step of gene expression in which a particular segment of DNA is copied into RNA (mRNA tRNA or rRNA) by the enzyme RNA polymerase
Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the
expression of one or more genes by binding to the operator or associated silencers
Transcription
Arc molecule
bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally
defined by their ability to be transcribed in the presence of protein synthesis inhibitors
Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are
short chains of amino acid monomers linked by peptide (amide) bonds
bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Levels of protein structure
1 Primary ndash amino acid sequence and disulfide bonds2 Secondary ndash Local folding to form regular elements3 Tertiary ndash 3 Dimensional folded structure4 Quaternary ndash Tertiary structure of multimeric proteins
Alpha Helix
bull It is a secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( hydrogen bonding)
Beta Sheet
bull The β sheet (also β-pleated sheet) is the second form of regular secondary structure in proteins bull Beta sheets consist of beta strands connected
laterally by at least two or three backbone hydrogen bonds forming a generally twisted pleated sheetbull A beta strand (also β strand) is a stretch of
polypeptide chain typically 3 to 10 amino acids long with backbone in an extended conformation
Beta sheet
Operatorbull In genetics an operator is a segment of DNA to which a
transcription factor binds to regulate gene expression The transcription factor is typically a repressor which can bind to the operator to prevent transcription
bull Transcription is the first step of gene expression in which a particular segment of DNA is copied into RNA (mRNA tRNA or rRNA) by the enzyme RNA polymerase
Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the
expression of one or more genes by binding to the operator or associated silencers
Transcription
Arc molecule
bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally
defined by their ability to be transcribed in the presence of protein synthesis inhibitors
Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are
short chains of amino acid monomers linked by peptide (amide) bonds
bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Alpha Helix
bull It is a secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( hydrogen bonding)
Beta Sheet
bull The β sheet (also β-pleated sheet) is the second form of regular secondary structure in proteins bull Beta sheets consist of beta strands connected
laterally by at least two or three backbone hydrogen bonds forming a generally twisted pleated sheetbull A beta strand (also β strand) is a stretch of
polypeptide chain typically 3 to 10 amino acids long with backbone in an extended conformation
Beta sheet
Operatorbull In genetics an operator is a segment of DNA to which a
transcription factor binds to regulate gene expression The transcription factor is typically a repressor which can bind to the operator to prevent transcription
bull Transcription is the first step of gene expression in which a particular segment of DNA is copied into RNA (mRNA tRNA or rRNA) by the enzyme RNA polymerase
Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the
expression of one or more genes by binding to the operator or associated silencers
Transcription
Arc molecule
bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally
defined by their ability to be transcribed in the presence of protein synthesis inhibitors
Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are
short chains of amino acid monomers linked by peptide (amide) bonds
bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Beta Sheet
bull The β sheet (also β-pleated sheet) is the second form of regular secondary structure in proteins bull Beta sheets consist of beta strands connected
laterally by at least two or three backbone hydrogen bonds forming a generally twisted pleated sheetbull A beta strand (also β strand) is a stretch of
polypeptide chain typically 3 to 10 amino acids long with backbone in an extended conformation
Beta sheet
Operatorbull In genetics an operator is a segment of DNA to which a
transcription factor binds to regulate gene expression The transcription factor is typically a repressor which can bind to the operator to prevent transcription
bull Transcription is the first step of gene expression in which a particular segment of DNA is copied into RNA (mRNA tRNA or rRNA) by the enzyme RNA polymerase
Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the
expression of one or more genes by binding to the operator or associated silencers
Transcription
Arc molecule
bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally
defined by their ability to be transcribed in the presence of protein synthesis inhibitors
Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are
short chains of amino acid monomers linked by peptide (amide) bonds
bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Beta sheet
Operatorbull In genetics an operator is a segment of DNA to which a
transcription factor binds to regulate gene expression The transcription factor is typically a repressor which can bind to the operator to prevent transcription
bull Transcription is the first step of gene expression in which a particular segment of DNA is copied into RNA (mRNA tRNA or rRNA) by the enzyme RNA polymerase
Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the
expression of one or more genes by binding to the operator or associated silencers
Transcription
Arc molecule
bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally
defined by their ability to be transcribed in the presence of protein synthesis inhibitors
Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are
short chains of amino acid monomers linked by peptide (amide) bonds
bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Operatorbull In genetics an operator is a segment of DNA to which a
transcription factor binds to regulate gene expression The transcription factor is typically a repressor which can bind to the operator to prevent transcription
bull Transcription is the first step of gene expression in which a particular segment of DNA is copied into RNA (mRNA tRNA or rRNA) by the enzyme RNA polymerase
Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the
expression of one or more genes by binding to the operator or associated silencers
Transcription
Arc molecule
bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally
defined by their ability to be transcribed in the presence of protein synthesis inhibitors
Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are
short chains of amino acid monomers linked by peptide (amide) bonds
bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Repressorbull A repressor is a DNA- or RNA-binding protein that inhibits the
expression of one or more genes by binding to the operator or associated silencers
Transcription
Arc molecule
bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally
defined by their ability to be transcribed in the presence of protein synthesis inhibitors
Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are
short chains of amino acid monomers linked by peptide (amide) bonds
bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Transcription
Arc molecule
bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally
defined by their ability to be transcribed in the presence of protein synthesis inhibitors
Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are
short chains of amino acid monomers linked by peptide (amide) bonds
bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Arc molecule
bull Arc is a member of the immediate-early gene (IEG) familybull It is a rapidly activated class of genes functionally
defined by their ability to be transcribed in the presence of protein synthesis inhibitors
Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are
short chains of amino acid monomers linked by peptide (amide) bonds
bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Homodimerbull A protein composed of two identical polypeptide chainsbull Polypeptides are chains of amino acidsbull Peptides are naturally occurring biological molecules They are
short chains of amino acid monomers linked by peptide (amide) bonds
bull A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Objective
bull Design a single-chain variant of the Arc repressor homodimer bull Here1113088 strands that contact operator DNA are
connected by a hairpin turnbull The1113088 helices that form the tetrahelical scaffold of
the dimer are attached by a short linkerbull The designed protein represents a noncyclic
permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc) in which the two subunits are fused by a single linker which is very stable
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Arc repressor binding to DNA
Beta sheets in contact with operator DNA
Tetrahelical bundle
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Objectivebull The crystal structure of the permuted protein reveals an
essentially wild-type fold demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure
bull Wild type (abbreviation wt) refers to the phenotype of the typical form of a species as it occurs in nature
bull A phenotype is the composite of an organisms observable characteristics or traits such as its morphology development biochemical or physiological properties phenology behavior and products of behavior (such as a birds nest)
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Introduction
bull Consider a protein in which the 1113088helices and 1113088strands are disconnected but otherwise arranged properly in three dimensions so that the hydrophobic core and most native interactions remain intact
bull Hydrophobic ndash water ldquofearingrdquo
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Introduction
bull There are probably many ways to connect the secondary structure elements that are compatible with stable folding
bull However to our knowledge no successful examples of noncyclic permutations of protein structural elements have been reported ( in 2004) (Now 2 papers have been published one in 2005 another in 2006)
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Methods
bull Molecular Biologybull Constructed in a plasmid system by using a
combination of PCR amplification of portions of the arc-st11 gene from pSA700bull The structure of the final construct was
verified by DNA sequencing
PCR ndash Polymerase chain reaction-used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude generating millions of copies
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Methodsbull Protein Purification bull Uses Ecoli for growingbull Isopropyl 1113088-D-thiogalactoside was added and cells
were harvested after an additional 2 h of growth bull Processed using HCL sodium phosphate and glycerolbull The concentration of pArc was determined by
absorbance using an extinction coefficientbull Protein purification is a series of processes intended to
isolate one or a few proteins from a complex mixture usually cells tissues or whole organisms
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Methodsbull Crystallographybull Crystals of pArc or selenomethionine- substituted pArc
were grown at 20degC by hanging-drop vapor diffusion bull Finally cycles of refinements are done to get
isomorphous cell of the native crystal
Isomorphous ndash having same crystalline form
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Result - Structure
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Resultsbull Design As shown in Fig 1C the order of structural elements
in single-chain Arc-L1-Arc is arm-1113088-1113088A-1113088B-linker-arm-1113088- 1113088A-1113088B (17) We reengineered the sequence to encode a permuted single-chain protein (pArc) with structural elements rearranged in the order arm-1113088-linker-1113088-1113088A-1113088B-linker-1113088A-1113088B (Fig 1C) In pArc unlike Arc-L1-Arc the 1113088 strands that contact operator DNA are now positioned together in the N-terminal part of the single-chain molecule whereas the C-terminal portion comprises the tetra helical scaffold
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Results
bull Expression and Structure bull The pArc protein was expressed in E coli bull It was soluble bull It was well behaved during purification
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Results
bull Stability and Folding Kinetics bull Rearranging the order of secondary structural
elements in pArc affected neither global stability nor the ability of the permuted protein to fold extremely rapidly to its native conformation
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Results - Analytical Ultracentrifuge
bull X-axis is called radial distance Itrsquos a distance by which tubes are separated on centrifuge machines Varying distance = varying force
bull Y axix is absorbance through spectrophotometer (measures density) -gt corresponds to folding state of protein
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Results - Denaturation
bull Used denaturing agent to unfold protein and unfolding stability of two Arc s is shown similar
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Discussionbull Proteins are generally remarkably robust to amino acid
substitutions cyclic permutations insertions and even small deletions
bull The studies reported here add noncyclic permutation to the list of seemingly abusive sequence manipulations that proteins can tolerate
bull However not all cyclic permutations are tolerated and it seems likely that noncyclic permutations will be accepted at somewhat lower frequencies because the sequence changes and linker additions represent more dramatic alterations
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Discussionbull The Arc fold was maintained after noncyclic rearrangement of
its secondary structural elements which required the addition of two non-natural linkers
bull It was also found that the thermodynamic stability of the designed protein was similar to the Arc-L1-Arc
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Discussionbull Previous studies did not find noncyclic permutants of barnase
that folded like the wild-type protein but they made no attempt to link permuted structural elements in a fashion compatible with wild type folding
bull The protein design success mainly depends on the fold of the protein the topology and the required linkers
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Referencebull Tabtiang Ramon K Brent O Cezairliyan Robert A Grant Jesse C
Cochrane and Robert T Sauer Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure Proceedings of the National Academy of Sciences of the United States of America 102 no 7 (2005) 2305-2309
bull Tsuji Toru Kenji Yoshida Akira Satoh Toshiyuki Kohno Kensei Kobayashi and Hiroshi Yanagawa Foldability of barnase mutants obtained by permutation of modules or secondary structure units Journal of molecular biology 286 no 5 (1999) 1581-1596
bull Lee Cheolju Michael P Schwartz Sumit Prakash Masahiro Iwakura and Andreas Matouschek ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal Molecular cell 7 no 3 (2001) 627-637
bull Wikipedia
Questions
Questions