1

Proteins

Protein folding

10112010

Polypeptides and proteins 1

bull peptide short polymers of amino acidslinked by peptide bonds (lt ~50 amino acids)

bull protein long polymers of amino acidslinked by peptide bonds (gt ~50 amino acids)

Amino acid

bull A group of organic molecules that contains a basic amino group (-NH2) an acidic carboxyl group (-COOH) and an organic R group (or side chain) which is unique to each amino acid

Amino acidsbull The unit within a protein molecule

bull 20 amino acids building up the proteins within the human body

bull Essential amino acids (9 pcs) the human body can not poduce them in a proper amount (methionine)

bull To build a 100 amino acid long polymer from the 20 amino acids ndash the number of the variation is huge (20100

= ~ 1310130)

bull The order of the amio acids = amino acid sequence rarrprimary structure

Polypeptides and proteins 2

H2N ndash C ndash C

H

R1

O

OHH2N ndash C ndash C

H

R2

O

OH

amino-group carboxylic-group amino-group carboxylic-group

N ndash C ndash C

H

R2

O

OHH

H2N ndash C ndash C

H

R1

O

+ H2O

Dehydration or condensation

Amide

Peptide bondN-terminus C-terminus

Protein

1926 - James B Sumner (biochemist - USA) chrystallized an

enzyme called urease and showed that it is a protein rarr Nobel

prize in Chemistry (1946 - John Howard Northrop amp Wendell

Meredith Stanley)

1958 - Max Perutz amp Sir John Cowdery Kendrew solved the strucure of hemoglobin and myoglobin rarr Nobel prize in

Chemistry in 1962

(X-ray chrystallography rarr 3D structure)

2

bull structural (collagen)

bull transport (myosin)

bull biochemical reactions (enzyme)

bull immunology (antibodies)

bull signal transduction (hormones)

Protein functions Primary structure

The sequence of the monomeric units

within a chain of amino-acids connected by peptide bounds

Primary structure

C

N

O

C

Primary structure

Forming a spatial-structure (structure in space)Motifs bull alpha helix

bull beta sheet

Secondary structure Alpha helix

H

O

H O

Hydrogen-bond

Rolling up on the surface of an imaginary cylinder (+ or -)

3

Hydrogen bond

An electrostatic dipole-dipole interactionthat involves a hydrogen atom

δ+ δ+

δ-

δ+ δ+

δ-

Electronegativity a chemical property that describes the ability of an atom to attract electrons towards itself

H2O

H2O

Beta sheet

Parallel and antiparallel beta sheets

Tertiary structure

bull Folding Forming the final functional 3D forms of the proteins

bull Domain formation

bull Under physiological conditions a spontaneos disorder hArrorder transition = bdquofoldingrdquo

bull Chaperon A guide or companion to the protein that help to form its tertiary structure

bull Disulfide bridge hidrogen bond and hydrophobicinteractions are sabilizing the folded protein

Disulfide bond

A covalent bound (primary) forming between thiol groups (eg cysteine)

A link between two sulfur atoms

Hydrophobic interactions

bull No affinity for water (tending not to be dissolved in or mixed with water)

bull Usually non polar molecules are involved

bull one of the principal driving forces behind the protein folding

bull minimizing the number of hydrophobic side-chains exposed to water

bull the hydrophobic amino acids are shielded from the aqueous solvent

bull Very hydrophobic amino acids

Valine isoleucine isoleucine leucine methionine phenylalanine cysteine tryptophan

4

Tertiary structure Quaternary structure

The entire protein assembly

Two or more peptide chains forming the functinal form of a protein (eg hemoglobin)

wwwpdborg

Folding

A process where the tertiary structure (3D

shape) of a protein is formedThe functional form of the protein comes to

life

Misfolding

The folding is not succesfull (eg beta sheets instead of alpha helices) rarr misfolded proteins

The cell remove the wrong protein rarr the amount of the functional proteins decrease

The cell will not remove it rarr deposits (plaque) within the cells (Alzheimer disease)

Protein folding diseases

P53cancer

crystallinsCataract

rhodopsinRetinitis pigmentosa

Transthyretin lysosymeFamiliai amyloidoses

Prion proteinCreutzfeldt-Jakob disease

α-synucleinParkinsonrsquos disease

Amyloid β-peptidetauAlzheimerrsquos disease

CollagenScurvy

β-hexosaminidaseTay-Sachs disease

α1-Antitrypsin α1-Antitrypsin deficiency

HaemoglobinSickle cell anaemia

ProcollagenOsteogenesis imperfecta

FibrillinMarfan syndrome

HuntingtinHuntingtonrsquos disease

Phenylalanine hydroxilasephenylketonuria

Cystic fibrosis trans-membran regulatorCystic fibrosis

Low-density lipoprtotein receptorHypercholesterolaemia

PROTEINDISEASE

Christian Boehmer Anfinsen (biochemist-USA) March 26 1916 Monessen Pa USA - May 14 1995

Randallstown Md

Theories behind the Folding

5

Anfinsen experiment I

- SS -

- SS -

- SS -

- SS -- SS -

- SS -

RNase A

SH -SH -

- SH

- SH

- SH

- SH

Unfolded

(unstructured)

protein without

functional activity

Removing the denaturing agents rarr folded structured functional protein

Denaturation with

8M urea

β-mercaptoethanol

Interpretation thermodynamic hypothesis ndash under physiological conditions the native form of the protein tends to achieve a minimum in Gibbs free energy

Conlusion The proteins can fold spontaneously

The 3D structure of the proteins is encoded within their primary structure

rarr 1972 Nobel price in chemistry (Stanford Moore amp William H Stein)

Anfinsen experiment II

The energy landscape theories

1 Levinthalrsquos pradox

2 The folding funnel

The energy landscape theories

Conformationaldistribution

en

erg

y

A model describes the relationship between the different conformations and energy levels

Central hole (cavity) for the final

(functional) state of the protein

Levinthalrsquos paradox

1968 - Cyrus Levinthal very large number

of degrees of freedom in an unfolded

polypeptide chain rarr the number of the

possible conformations is huge

Is the protein sampling all the possible

conformations

conformation

en

erg

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

Levinthalrsquos paradox

conformation

en

erg

y

Calculation Forming a protein that contains 25 bonds and all bound can be in 5

different conformations

n=5

i=25

N=ni rarr 525

The length of forming one conformation 1 ns

(10-9s)

The length of trying all the possible

conformations rarr 52510-9 s = 298109s =

~95 year harr micromicromicromicros - ms

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

6

Levinthalrsquos paradox

conformation

en

erg

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

Conclusions1 an intensive purely random search

cannot succeed

2 the native state is achieved through

a directed search

The folding funnel

conformation

Energ

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

bull Large number of folding path with equal

probability ( harr one folding path in Levinthalrsquos idea)

bull All paths lead directly to the native state

(energetic minimum)

bull The depth of the well symbolize the energetic

stabilization of the native state versus the

denatured state

bull The width of the well symbolize the entropy of the

system

bull The surface outside the well can symbolize the heterogeneity of the random coil state

bull directed search

Thermal fluctuation of the chemical bonds (20degC)

bull Covalent-bond 2-10 eV

rarr n1n

0~ 138times10-85

bull H-bond 005-03 eV

rarr n1n

0~ 0005-16

bull Van der Waals bond lt 0025eV

rarr n1n

0~ 368

bull Dipole-dipole interaction~ 00125-005 eV

rarr n1n

0~ 135-60

Binding energy the net energy required to decompose a molecule to break up chemical bonds

Main stabilizing

forces within the

macromolecules

Conformational dynamics

bull Continuous conformational transitions within the macromolecular structures

bull Martin Karplus ndash 1986

Haemoglobin ndash structure from x-ray diffraction

+ O2

Flexibility and protein function

bull Proteins can adapt to their ligand (larrkey-lock)

ndash induced fit during the ligand binding both the

ligand and the protein can adjust its structure to the

presence of the other

bull Protein flexibility is necessary for their

biochemical function

The end

2

bull structural (collagen)

bull transport (myosin)

bull biochemical reactions (enzyme)

bull immunology (antibodies)

bull signal transduction (hormones)

Protein functions Primary structure

The sequence of the monomeric units

within a chain of amino-acids connected by peptide bounds

Primary structure

C

N

O

C

Primary structure

Forming a spatial-structure (structure in space)Motifs bull alpha helix

bull beta sheet

Secondary structure Alpha helix

H

O

H O

Hydrogen-bond

Rolling up on the surface of an imaginary cylinder (+ or -)

3

Hydrogen bond

An electrostatic dipole-dipole interactionthat involves a hydrogen atom

δ+ δ+

δ-

δ+ δ+

δ-

Electronegativity a chemical property that describes the ability of an atom to attract electrons towards itself

H2O

H2O

Beta sheet

Parallel and antiparallel beta sheets

Tertiary structure

bull Folding Forming the final functional 3D forms of the proteins

bull Domain formation

bull Under physiological conditions a spontaneos disorder hArrorder transition = bdquofoldingrdquo

bull Chaperon A guide or companion to the protein that help to form its tertiary structure

bull Disulfide bridge hidrogen bond and hydrophobicinteractions are sabilizing the folded protein

Disulfide bond

A covalent bound (primary) forming between thiol groups (eg cysteine)

A link between two sulfur atoms

Hydrophobic interactions

bull No affinity for water (tending not to be dissolved in or mixed with water)

bull Usually non polar molecules are involved

bull one of the principal driving forces behind the protein folding

bull minimizing the number of hydrophobic side-chains exposed to water

bull the hydrophobic amino acids are shielded from the aqueous solvent

bull Very hydrophobic amino acids

Valine isoleucine isoleucine leucine methionine phenylalanine cysteine tryptophan

4

Tertiary structure Quaternary structure

The entire protein assembly

Two or more peptide chains forming the functinal form of a protein (eg hemoglobin)

wwwpdborg

Folding

A process where the tertiary structure (3D

shape) of a protein is formedThe functional form of the protein comes to

life

Misfolding

The folding is not succesfull (eg beta sheets instead of alpha helices) rarr misfolded proteins

The cell remove the wrong protein rarr the amount of the functional proteins decrease

The cell will not remove it rarr deposits (plaque) within the cells (Alzheimer disease)

Protein folding diseases

P53cancer

crystallinsCataract

rhodopsinRetinitis pigmentosa

Transthyretin lysosymeFamiliai amyloidoses

Prion proteinCreutzfeldt-Jakob disease

α-synucleinParkinsonrsquos disease

Amyloid β-peptidetauAlzheimerrsquos disease

CollagenScurvy

β-hexosaminidaseTay-Sachs disease

α1-Antitrypsin α1-Antitrypsin deficiency

HaemoglobinSickle cell anaemia

ProcollagenOsteogenesis imperfecta

FibrillinMarfan syndrome

HuntingtinHuntingtonrsquos disease

Phenylalanine hydroxilasephenylketonuria

Cystic fibrosis trans-membran regulatorCystic fibrosis

Low-density lipoprtotein receptorHypercholesterolaemia

PROTEINDISEASE

Christian Boehmer Anfinsen (biochemist-USA) March 26 1916 Monessen Pa USA - May 14 1995

Randallstown Md

Theories behind the Folding

5

Anfinsen experiment I

- SS -

- SS -

- SS -

- SS -- SS -

- SS -

RNase A

SH -SH -

- SH

- SH

- SH

- SH

Unfolded

(unstructured)

protein without

functional activity

Removing the denaturing agents rarr folded structured functional protein

Denaturation with

8M urea

β-mercaptoethanol

Interpretation thermodynamic hypothesis ndash under physiological conditions the native form of the protein tends to achieve a minimum in Gibbs free energy

Conlusion The proteins can fold spontaneously

The 3D structure of the proteins is encoded within their primary structure

rarr 1972 Nobel price in chemistry (Stanford Moore amp William H Stein)

Anfinsen experiment II

The energy landscape theories

1 Levinthalrsquos pradox

2 The folding funnel

The energy landscape theories

Conformationaldistribution

en

erg

y

A model describes the relationship between the different conformations and energy levels

Central hole (cavity) for the final

(functional) state of the protein

Levinthalrsquos paradox

1968 - Cyrus Levinthal very large number

of degrees of freedom in an unfolded

polypeptide chain rarr the number of the

possible conformations is huge

Is the protein sampling all the possible

conformations

conformation

en

erg

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

Levinthalrsquos paradox

conformation

en

erg

y

Calculation Forming a protein that contains 25 bonds and all bound can be in 5

different conformations

n=5

i=25

N=ni rarr 525

The length of forming one conformation 1 ns

(10-9s)

The length of trying all the possible

conformations rarr 52510-9 s = 298109s =

~95 year harr micromicromicromicros - ms

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

6

Levinthalrsquos paradox

conformation

en

erg

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

Conclusions1 an intensive purely random search

cannot succeed

2 the native state is achieved through

a directed search

The folding funnel

conformation

Energ

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

bull Large number of folding path with equal

probability ( harr one folding path in Levinthalrsquos idea)

bull All paths lead directly to the native state

(energetic minimum)

bull The depth of the well symbolize the energetic

stabilization of the native state versus the

denatured state

bull The width of the well symbolize the entropy of the

system

bull The surface outside the well can symbolize the heterogeneity of the random coil state

bull directed search

Thermal fluctuation of the chemical bonds (20degC)

bull Covalent-bond 2-10 eV

rarr n1n

0~ 138times10-85

bull H-bond 005-03 eV

rarr n1n

0~ 0005-16

bull Van der Waals bond lt 0025eV

rarr n1n

0~ 368

bull Dipole-dipole interaction~ 00125-005 eV

rarr n1n

0~ 135-60

Binding energy the net energy required to decompose a molecule to break up chemical bonds

Main stabilizing

forces within the

macromolecules

Conformational dynamics

bull Continuous conformational transitions within the macromolecular structures

bull Martin Karplus ndash 1986

Haemoglobin ndash structure from x-ray diffraction

+ O2

Flexibility and protein function

bull Proteins can adapt to their ligand (larrkey-lock)

ndash induced fit during the ligand binding both the

ligand and the protein can adjust its structure to the

presence of the other

bull Protein flexibility is necessary for their

biochemical function

The end

3

Hydrogen bond

An electrostatic dipole-dipole interactionthat involves a hydrogen atom

δ+ δ+

δ-

δ+ δ+

δ-

Electronegativity a chemical property that describes the ability of an atom to attract electrons towards itself

H2O

H2O

Beta sheet

Parallel and antiparallel beta sheets

Tertiary structure

bull Folding Forming the final functional 3D forms of the proteins

bull Domain formation

bull Under physiological conditions a spontaneos disorder hArrorder transition = bdquofoldingrdquo

bull Chaperon A guide or companion to the protein that help to form its tertiary structure

bull Disulfide bridge hidrogen bond and hydrophobicinteractions are sabilizing the folded protein

Disulfide bond

A covalent bound (primary) forming between thiol groups (eg cysteine)

A link between two sulfur atoms

Hydrophobic interactions

bull No affinity for water (tending not to be dissolved in or mixed with water)

bull Usually non polar molecules are involved

bull one of the principal driving forces behind the protein folding

bull minimizing the number of hydrophobic side-chains exposed to water

bull the hydrophobic amino acids are shielded from the aqueous solvent

bull Very hydrophobic amino acids

Valine isoleucine isoleucine leucine methionine phenylalanine cysteine tryptophan

4

Tertiary structure Quaternary structure

The entire protein assembly

Two or more peptide chains forming the functinal form of a protein (eg hemoglobin)

wwwpdborg

Folding

A process where the tertiary structure (3D

shape) of a protein is formedThe functional form of the protein comes to

life

Misfolding

The folding is not succesfull (eg beta sheets instead of alpha helices) rarr misfolded proteins

The cell remove the wrong protein rarr the amount of the functional proteins decrease

The cell will not remove it rarr deposits (plaque) within the cells (Alzheimer disease)

Protein folding diseases

P53cancer

crystallinsCataract

rhodopsinRetinitis pigmentosa

Transthyretin lysosymeFamiliai amyloidoses

Prion proteinCreutzfeldt-Jakob disease

α-synucleinParkinsonrsquos disease

Amyloid β-peptidetauAlzheimerrsquos disease

CollagenScurvy

β-hexosaminidaseTay-Sachs disease

α1-Antitrypsin α1-Antitrypsin deficiency

HaemoglobinSickle cell anaemia

ProcollagenOsteogenesis imperfecta

FibrillinMarfan syndrome

HuntingtinHuntingtonrsquos disease

Phenylalanine hydroxilasephenylketonuria

Cystic fibrosis trans-membran regulatorCystic fibrosis

Low-density lipoprtotein receptorHypercholesterolaemia

PROTEINDISEASE

Christian Boehmer Anfinsen (biochemist-USA) March 26 1916 Monessen Pa USA - May 14 1995

Randallstown Md

Theories behind the Folding

5

Anfinsen experiment I

- SS -

- SS -

- SS -

- SS -- SS -

- SS -

RNase A

SH -SH -

- SH

- SH

- SH

- SH

Unfolded

(unstructured)

protein without

functional activity

Removing the denaturing agents rarr folded structured functional protein

Denaturation with

8M urea

β-mercaptoethanol

Interpretation thermodynamic hypothesis ndash under physiological conditions the native form of the protein tends to achieve a minimum in Gibbs free energy

Conlusion The proteins can fold spontaneously

The 3D structure of the proteins is encoded within their primary structure

rarr 1972 Nobel price in chemistry (Stanford Moore amp William H Stein)

Anfinsen experiment II

The energy landscape theories

1 Levinthalrsquos pradox

2 The folding funnel

The energy landscape theories

Conformationaldistribution

en

erg

y

A model describes the relationship between the different conformations and energy levels

Central hole (cavity) for the final

(functional) state of the protein

Levinthalrsquos paradox

1968 - Cyrus Levinthal very large number

of degrees of freedom in an unfolded

polypeptide chain rarr the number of the

possible conformations is huge

Is the protein sampling all the possible

conformations

conformation

en

erg

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

Levinthalrsquos paradox

conformation

en

erg

y

Calculation Forming a protein that contains 25 bonds and all bound can be in 5

different conformations

n=5

i=25

N=ni rarr 525

The length of forming one conformation 1 ns

(10-9s)

The length of trying all the possible

conformations rarr 52510-9 s = 298109s =

~95 year harr micromicromicromicros - ms

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

6

Levinthalrsquos paradox

conformation

en

erg

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

Conclusions1 an intensive purely random search

cannot succeed

2 the native state is achieved through

a directed search

The folding funnel

conformation

Energ

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

bull Large number of folding path with equal

probability ( harr one folding path in Levinthalrsquos idea)

bull All paths lead directly to the native state

(energetic minimum)

bull The depth of the well symbolize the energetic

stabilization of the native state versus the

denatured state

bull The width of the well symbolize the entropy of the

system

bull The surface outside the well can symbolize the heterogeneity of the random coil state

bull directed search

Thermal fluctuation of the chemical bonds (20degC)

bull Covalent-bond 2-10 eV

rarr n1n

0~ 138times10-85

bull H-bond 005-03 eV

rarr n1n

0~ 0005-16

bull Van der Waals bond lt 0025eV

rarr n1n

0~ 368

bull Dipole-dipole interaction~ 00125-005 eV

rarr n1n

0~ 135-60

Binding energy the net energy required to decompose a molecule to break up chemical bonds

Main stabilizing

forces within the

macromolecules

Conformational dynamics

bull Continuous conformational transitions within the macromolecular structures

bull Martin Karplus ndash 1986

Haemoglobin ndash structure from x-ray diffraction

+ O2

Flexibility and protein function

bull Proteins can adapt to their ligand (larrkey-lock)

ndash induced fit during the ligand binding both the

ligand and the protein can adjust its structure to the

presence of the other

bull Protein flexibility is necessary for their

biochemical function

The end

4

Tertiary structure Quaternary structure

The entire protein assembly

Two or more peptide chains forming the functinal form of a protein (eg hemoglobin)

wwwpdborg

Folding

A process where the tertiary structure (3D

shape) of a protein is formedThe functional form of the protein comes to

life

Misfolding

The folding is not succesfull (eg beta sheets instead of alpha helices) rarr misfolded proteins

The cell remove the wrong protein rarr the amount of the functional proteins decrease

The cell will not remove it rarr deposits (plaque) within the cells (Alzheimer disease)

Protein folding diseases

P53cancer

crystallinsCataract

rhodopsinRetinitis pigmentosa

Transthyretin lysosymeFamiliai amyloidoses

Prion proteinCreutzfeldt-Jakob disease

α-synucleinParkinsonrsquos disease

Amyloid β-peptidetauAlzheimerrsquos disease

CollagenScurvy

β-hexosaminidaseTay-Sachs disease

α1-Antitrypsin α1-Antitrypsin deficiency

HaemoglobinSickle cell anaemia

ProcollagenOsteogenesis imperfecta

FibrillinMarfan syndrome

HuntingtinHuntingtonrsquos disease

Phenylalanine hydroxilasephenylketonuria

Cystic fibrosis trans-membran regulatorCystic fibrosis

Low-density lipoprtotein receptorHypercholesterolaemia

PROTEINDISEASE

Christian Boehmer Anfinsen (biochemist-USA) March 26 1916 Monessen Pa USA - May 14 1995

Randallstown Md

Theories behind the Folding

5

Anfinsen experiment I

- SS -

- SS -

- SS -

- SS -- SS -

- SS -

RNase A

SH -SH -

- SH

- SH

- SH

- SH

Unfolded

(unstructured)

protein without

functional activity

Removing the denaturing agents rarr folded structured functional protein

Denaturation with

8M urea

β-mercaptoethanol

Interpretation thermodynamic hypothesis ndash under physiological conditions the native form of the protein tends to achieve a minimum in Gibbs free energy

Conlusion The proteins can fold spontaneously

The 3D structure of the proteins is encoded within their primary structure

rarr 1972 Nobel price in chemistry (Stanford Moore amp William H Stein)

Anfinsen experiment II

The energy landscape theories

1 Levinthalrsquos pradox

2 The folding funnel

The energy landscape theories

Conformationaldistribution

en

erg

y

A model describes the relationship between the different conformations and energy levels

Central hole (cavity) for the final

(functional) state of the protein

Levinthalrsquos paradox

1968 - Cyrus Levinthal very large number

of degrees of freedom in an unfolded

polypeptide chain rarr the number of the

possible conformations is huge

Is the protein sampling all the possible

conformations

conformation

en

erg

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

Levinthalrsquos paradox

conformation

en

erg

y

Calculation Forming a protein that contains 25 bonds and all bound can be in 5

different conformations

n=5

i=25

N=ni rarr 525

The length of forming one conformation 1 ns

(10-9s)

The length of trying all the possible

conformations rarr 52510-9 s = 298109s =

~95 year harr micromicromicromicros - ms

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

6

Levinthalrsquos paradox

conformation

en

erg

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

Conclusions1 an intensive purely random search

cannot succeed

2 the native state is achieved through

a directed search

The folding funnel

conformation

Energ

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

bull Large number of folding path with equal

probability ( harr one folding path in Levinthalrsquos idea)

bull All paths lead directly to the native state

(energetic minimum)

bull The depth of the well symbolize the energetic

stabilization of the native state versus the

denatured state

bull The width of the well symbolize the entropy of the

system

bull The surface outside the well can symbolize the heterogeneity of the random coil state

bull directed search

Thermal fluctuation of the chemical bonds (20degC)

bull Covalent-bond 2-10 eV

rarr n1n

0~ 138times10-85

bull H-bond 005-03 eV

rarr n1n

0~ 0005-16

bull Van der Waals bond lt 0025eV

rarr n1n

0~ 368

bull Dipole-dipole interaction~ 00125-005 eV

rarr n1n

0~ 135-60

Binding energy the net energy required to decompose a molecule to break up chemical bonds

Main stabilizing

forces within the

macromolecules

Conformational dynamics

bull Continuous conformational transitions within the macromolecular structures

bull Martin Karplus ndash 1986

Haemoglobin ndash structure from x-ray diffraction

+ O2

Flexibility and protein function

bull Proteins can adapt to their ligand (larrkey-lock)

ndash induced fit during the ligand binding both the

ligand and the protein can adjust its structure to the

presence of the other

bull Protein flexibility is necessary for their

biochemical function

The end

5

Anfinsen experiment I

- SS -

- SS -

- SS -

- SS -- SS -

- SS -

RNase A

SH -SH -

- SH

- SH

- SH

- SH

Unfolded

(unstructured)

protein without

functional activity

Removing the denaturing agents rarr folded structured functional protein

Denaturation with

8M urea

β-mercaptoethanol

Interpretation thermodynamic hypothesis ndash under physiological conditions the native form of the protein tends to achieve a minimum in Gibbs free energy

Conlusion The proteins can fold spontaneously

The 3D structure of the proteins is encoded within their primary structure

rarr 1972 Nobel price in chemistry (Stanford Moore amp William H Stein)

Anfinsen experiment II

The energy landscape theories

1 Levinthalrsquos pradox

2 The folding funnel

The energy landscape theories

Conformationaldistribution

en

erg

y

A model describes the relationship between the different conformations and energy levels

Central hole (cavity) for the final

(functional) state of the protein

Levinthalrsquos paradox

1968 - Cyrus Levinthal very large number

of degrees of freedom in an unfolded

polypeptide chain rarr the number of the

possible conformations is huge

Is the protein sampling all the possible

conformations

conformation

en

erg

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

Levinthalrsquos paradox

conformation

en

erg

y

Calculation Forming a protein that contains 25 bonds and all bound can be in 5

different conformations

n=5

i=25

N=ni rarr 525

The length of forming one conformation 1 ns

(10-9s)

The length of trying all the possible

conformations rarr 52510-9 s = 298109s =

~95 year harr micromicromicromicros - ms

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

6

Levinthalrsquos paradox

conformation

en

erg

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

Conclusions1 an intensive purely random search

cannot succeed

2 the native state is achieved through

a directed search

The folding funnel

conformation

Energ

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

bull Large number of folding path with equal

probability ( harr one folding path in Levinthalrsquos idea)

bull All paths lead directly to the native state

(energetic minimum)

bull The depth of the well symbolize the energetic

stabilization of the native state versus the

denatured state

bull The width of the well symbolize the entropy of the

system

bull The surface outside the well can symbolize the heterogeneity of the random coil state

bull directed search

Thermal fluctuation of the chemical bonds (20degC)

bull Covalent-bond 2-10 eV

rarr n1n

0~ 138times10-85

bull H-bond 005-03 eV

rarr n1n

0~ 0005-16

bull Van der Waals bond lt 0025eV

rarr n1n

0~ 368

bull Dipole-dipole interaction~ 00125-005 eV

rarr n1n

0~ 135-60

Binding energy the net energy required to decompose a molecule to break up chemical bonds

Main stabilizing

forces within the

macromolecules

Conformational dynamics

bull Continuous conformational transitions within the macromolecular structures

bull Martin Karplus ndash 1986

Haemoglobin ndash structure from x-ray diffraction

+ O2

Flexibility and protein function

bull Proteins can adapt to their ligand (larrkey-lock)

ndash induced fit during the ligand binding both the

ligand and the protein can adjust its structure to the

presence of the other

bull Protein flexibility is necessary for their

biochemical function

The end

6

Levinthalrsquos paradox

conformation

en

erg

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

Conclusions1 an intensive purely random search

cannot succeed

2 the native state is achieved through

a directed search

The folding funnel

conformation

Energ

y

Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9

bull Large number of folding path with equal

probability ( harr one folding path in Levinthalrsquos idea)

bull All paths lead directly to the native state

(energetic minimum)

bull The depth of the well symbolize the energetic

stabilization of the native state versus the

denatured state

bull The width of the well symbolize the entropy of the

system

bull The surface outside the well can symbolize the heterogeneity of the random coil state

bull directed search

Thermal fluctuation of the chemical bonds (20degC)

bull Covalent-bond 2-10 eV

rarr n1n

0~ 138times10-85

bull H-bond 005-03 eV

rarr n1n

0~ 0005-16

bull Van der Waals bond lt 0025eV

rarr n1n

0~ 368

bull Dipole-dipole interaction~ 00125-005 eV

rarr n1n

0~ 135-60

Binding energy the net energy required to decompose a molecule to break up chemical bonds

Main stabilizing

forces within the

macromolecules

Conformational dynamics

bull Continuous conformational transitions within the macromolecular structures

bull Martin Karplus ndash 1986

Haemoglobin ndash structure from x-ray diffraction

+ O2

Flexibility and protein function

bull Proteins can adapt to their ligand (larrkey-lock)

ndash induced fit during the ligand binding both the

ligand and the protein can adjust its structure to the

presence of the other

bull Protein flexibility is necessary for their

biochemical function

The end