Chapter - 2Chapter - 2

Structure of Structure of ProteinsProteins

Lecture 4Lecture 4ContentsContents

OverviewOverview

Primary structure of proteins:Primary structure of proteins:– Peptide bondPeptide bond

Secondary structure of proteins:Secondary structure of proteins:– - Helix- Helix– - Sheet- Sheet– - Bends (reverse turns)- Bends (reverse turns)

OVERVIEWOVERVIEW

– The The 20 amino acids20 amino acids commonly found in commonly found in proteins are proteins are joined togetherjoined together by by peptide peptide bonds. bonds.

– The The linear sequence of the linked aminolinear sequence of the linked amino acids acids containscontains the information necessary the information necessary to generate a protein molecule with a to generate a protein molecule with a unique three-dimensional shape. unique three-dimensional shape.

– The The complexity of protein structurecomplexity of protein structure is best is best analyzed byanalyzed by considering the molecule in considering the molecule in terms of four organizational levels, terms of four organizational levels, namely: namely: primaryprimary, , secondarysecondary, , tertiarytertiary, and , and quaternaryquaternary (Figure 2.1).(Figure 2.1).

– An An examination of these hierarchies of examination of these hierarchies of increasing complexityincreasing complexity has has revealed thatrevealed that certain structural elements are repeated in certain structural elements are repeated in a wide variety of proteinsa wide variety of proteins, suggesting that , suggesting that there are general "rules" regarding the there are general "rules" regarding the ways in which proteins fold. ways in which proteins fold.

– These These repeated structural elementsrepeated structural elements range range from from simple combinationssimple combinations of of -helices-helices and and -sheets-sheets small motifssmall motifs to the to the complex complex folding of polypeptide domainsfolding of polypeptide domains of of multifunctional proteins.multifunctional proteins.

II. PRIMARY STRUCTURE OF PROTEINSII. PRIMARY STRUCTURE OF PROTEINS

The sequence of amino acids in a protein is The sequence of amino acids in a protein is called called the primary structurethe primary structure of the protein. of the protein.

Understanding the primary structure of Understanding the primary structure of proteins is important because proteins is important because many genetic many genetic diseases result in proteins with abnormal diseases result in proteins with abnormal amino acid sequences amino acid sequences improper folding and improper folding and loss or impairment of normal function. loss or impairment of normal function.

If the primary structures of the normal and the If the primary structures of the normal and the mutated proteins are knownmutated proteins are known, , diagnosis or diagnosis or studying the disease.studying the disease.

A. Peptide bondA. Peptide bond

In In proteinsproteins, , amino acids are joined amino acids are joined covalently by covalently by peptide bondspeptide bonds, , ( which are ( which are amide linkages between the amide linkages between the -carboxyl -carboxyl group of one amino acid and the group of one amino acid and the -amino -amino group of another).group of another).

For example: For example: valinevaline and and alaninealanine form the form the dipeptide valylalaninedipeptide valylalanine through the formation through the formation of a peptide bond of a peptide bond (Figure 2.2).(Figure 2.2).

Peptide bondsPeptide bonds are are not broken by not broken by conditions that denature proteinsconditions that denature proteins, , (e.g. (e.g. heatingheating or or high concentrations of urea)high concentrations of urea)..

Nonenzymic Hydrolysis of these bondsNonenzymic Hydrolysis of these bonds requiresrequires prolonged exposure to a strong prolonged exposure to a strong

acid or baseacid or base at high temperatures at high temperatures . .

1. Naming the peptide: 1. Naming the peptide:

– By conventionBy convention, , the the free amino end of the peptide chainfree amino end of the peptide chain (N- (N-terminal) is terminal) is written to the leftwritten to the left

the the free carboxyl endfree carboxyl end (C-terminal) (C-terminal) to the rightto the right. .

– So, all amino sequences are read from the So, all amino sequences are read from the N- to the C-terminal end of the peptide. N- to the C-terminal end of the peptide.

– e.g. the e.g. the order of the amino acidsorder of the amino acids is is "valine, "valine, alanine"alanine" not not "alanine, valine“ "alanine, valine“ (Figure 2.2 A) (Figure 2.2 A)

– Linkage of many amino acids through Linkage of many amino acids through peptide bonds peptide bonds an unbranched chain an unbranched chain called a called a polypeptidepolypeptide..

– Each component amino acidEach component amino acid in a polypeptide in a polypeptide is called a is called a “residue”“residue” or or “moiety”.“moiety”.

– When a polypeptide is namedWhen a polypeptide is named, all amino acid , all amino acid residues have residues have their suffixestheir suffixes (—ine, —an, (—ine, —an, —ic, or —ate) —ic, or —ate) changed tochanged to —yl, —yl, with the with the exception ofexception of the C-terminal amino acid. the C-terminal amino acid.

– For exampleFor example, a tripeptide composed of an , a tripeptide composed of an N-terminalN-terminal ( (valinevaline& & glycineglycine) and a ) and a C-terminalC-terminal ((leucineleucine) is called ) is called valylglycylleucinevalylglycylleucine..

22 . .Characteristics of the peptide bondCharacteristics of the peptide bond::

The The peptide bondpeptide bond has a partial double- has a partial double-bond character, that is, it is bond character, that is, it is shorter shorter than a single bondthan a single bond, and is , and is rigidrigid and and planar planar (Figure 2.2B).(Figure 2.2B).

This This prevents free rotation around the prevents free rotation around the bondbond between the carbonyl carbon and between the carbonyl carbon and the nitrogen of the peptide bond.the nitrogen of the peptide bond.

However, However, the bonds between the the bonds between the -carbons -carbons and the and the -amino-amino or or -carboxyl groups-carboxyl groups can be can be freely rotatedfreely rotated (although they are limited by (although they are limited by the size and character of the R-groups).the size and character of the R-groups).

This allows the polypeptide chain to assume This allows the polypeptide chain to assume a variety of possible configurations. a variety of possible configurations.

The The peptide bondpeptide bond is generally is generally a trans bonda trans bond (instead of cis).(instead of cis). (Figure 2.2B),(Figure 2.2B), in large part in large part because of steric interference of the R-because of steric interference of the R-groups when in the cis position.groups when in the cis position.

33 . .Polarity of the peptide bondPolarity of the peptide bond::

Like all amide linkagesLike all amide linkages, the —C=O and —NH groups , the —C=O and —NH groups of the peptide bond are uncharged, and neither of the peptide bond are uncharged, and neither accept nor release protons over the pH range of 2 to accept nor release protons over the pH range of 2 to 12. 12.

The The charged groups present in polypeptidescharged groups present in polypeptides consist consist solely of the N-terminal solely of the N-terminal -amino group, the C--amino group, the C-terminal terminal -carboxyl group-carboxyl group and and any ionized groupsany ionized groups present in the side chains of the constituent amino present in the side chains of the constituent amino acids. acids.

[[TheThe —C=O and —NH groups —C=O and —NH groups of the peptide bond areof the peptide bond are polarpolar, , and areand are involved in hydrogen bondsinvolved in hydrogen bonds, , for for exampleexample, in , in -helices-helices and and -sheet-sheet structures]. structures].

III. SECONDARY STRUCTURE OF PROTEINSIII. SECONDARY STRUCTURE OF PROTEINS

The polypeptide backboneThe polypeptide backbone does not assume does not assume a random three-dimensional structurea random three-dimensional structure, but , but instead generally forms regular instead generally forms regular arrangements of amino acids that are located arrangements of amino acids that are located near to each other in the linear sequence. near to each other in the linear sequence.

These arrangements are termed the These arrangements are termed the secondary structure of the polypeptide.secondary structure of the polypeptide.

The The -helix-helix, , -sheet-sheet, and , and -bend-bend are are examples of examples of secondary structuressecondary structures frequently frequently encountered in proteins.encountered in proteins.

A. A. -Helix-Helix

Several different polypeptide helices found in Several different polypeptide helices found in naturenature, but the , but the -helix is the most common. -helix is the most common.

It is a It is a spiral structurespiral structure, , consisting ofconsisting of a a tightly tightly packed, coiled polypeptide backbone corepacked, coiled polypeptide backbone core, , with with thethe side chains of the component amino side chains of the component amino acids extending outwardacids extending outward from the central from the central axis axis to avoid interfering sterically with each to avoid interfering sterically with each otherother (Figure 2.6).(Figure 2.6).

A A very diverse group of proteins contains very diverse group of proteins contains --heliceshelices. . For exampleFor example, the , the keratinskeratins are a are a family of closely related, family of closely related, fibrous proteinsfibrous proteins whose structure is nearly entirely whose structure is nearly entirely -helical-helical. .

They are a major component of tissues such They are a major component of tissues such as as hairhair and and skin,skin, and their and their rigidity rigidity is is determined bydetermined by the number of the number of disulfide bondsdisulfide bonds between the constituent polypeptide chains.between the constituent polypeptide chains.

In contrast to In contrast to keratinkeratin, ,

myoglobin,myoglobin, whose structure is approximately whose structure is approximately eighty percent eighty percent -helical, is a -helical, is a globularglobular, , flexible flexible moleculemolecule..

11 . .Hydrogen bondsHydrogen bonds::

An An -helix-helix is is stabilized bystabilized by extensive extensive hydrogen bondinghydrogen bonding between the peptide-bond between the peptide-bond carbonyl oxygens and amide hydrogens that carbonyl oxygens and amide hydrogens that are part of the polypeptide backboneare part of the polypeptide backbone

(Figure 2.6).(Figure 2.6).

The The hydrogen bondshydrogen bonds extend up the extend up the spiralspiral from the carbonyl oxygen of one from the carbonyl oxygen of one peptide bond to the -NH- group of a peptide peptide bond to the -NH- group of a peptide linkage four residues ahead in the linkage four residues ahead in the polypeptide.polypeptide.

This ensures that This ensures that all but the first and all but the first and last peptide bond componentslast peptide bond components are are linked to each other throughlinked to each other through hydrogen hydrogen bonds. bonds.

Hydrogen bondsHydrogen bonds are are individually weakindividually weak, , butbut they collectively serve to stabilize they collectively serve to stabilize the helixthe helix..

22 . .Amino acids per turnAmino acids per turn::

Each turn of an Each turn of an -helix contains 3.6 -helix contains 3.6 amino acids. amino acids.

Thus, Thus, amino acid residues spaced amino acid residues spaced three or four apart in the primary three or four apart in the primary sequence are spatially close togethersequence are spatially close together when folded in the when folded in the -helix.-helix.

33 . .Amino acids that disrupt an Amino acids that disrupt an -helix-helix::

ProlineProline disrupts an disrupts an -helix-helix because its imino because its imino group is not geometrically compatible with group is not geometrically compatible with the right-handed spiral of the the right-handed spiral of the -helix. -helix.

Instead, Instead, it inserts a kink in the chainit inserts a kink in the chain, which , which interferes with the smooth, helical structure. interferes with the smooth, helical structure.

Large number of charged amino acidsLarge number of charged amino acids ((glutamateglutamate, , aspartateaspartate, , histidinehistidine, , lysinelysine, or , or argininearginine) ) disrupt the helix disrupt the helix by forming ionic by forming ionic bondsbonds, , oror by electrostatically repelling each by electrostatically repelling each other.other.

Amino acids with bulky side chainsAmino acids with bulky side chains, ,

such as such as tryptophantryptophan, , oror amino acidsamino acids, such as , such as valine valine or or isoleucineisoleucine, ( that branch at the , ( that branch at the -carbon, -carbon, the first carbon in the R-group, next to the first carbon in the R-group, next to the the -carbon) -carbon) can interfere with can interfere with formation of the formation of the -helix-helix if they are if they are present in large numbers.present in large numbers.

B. B. -Sheet-SheetThe The -sheet-sheet is another form of is another form of secondary secondary structurestructure in which all of the peptide bond in which all of the peptide bond components are involved in hydrogen components are involved in hydrogen bonding.bonding. (Figure 2.7A).(Figure 2.7A).

The The surfaces of surfaces of -sheets-sheets appear appear "pleated""pleated" and these structures are, therefore, often and these structures are, therefore, often called “ called “ - pleated sheets".- pleated sheets".

When illustrations are made of protein When illustrations are made of protein structure, structure, -strands-strands are often visualized as are often visualized as broad arrowsbroad arrows (Figure (Figure 2.7B).2.7B).

11 . .Comparison of a Comparison of a -sheet and an -sheet and an -helix-helix::

– Unlike the a-helixUnlike the a-helix, , -sheets-sheets are are composed of two or more peptide composed of two or more peptide chains chains ((-strands),-strands), or or segments of segments of polypeptide chains,polypeptide chains, which are which are almost fully extended.almost fully extended.

– in in -sheets-sheets the the hydrogen bonds are hydrogen bonds are perpendicular to the polypeptide perpendicular to the polypeptide backbonebackbone (Figure 2.7A).(Figure 2.7A).

22 . .Parallel and antiparallel sheetsParallel and antiparallel sheets::

A A -sheet-sheet can be can be formed fromformed from two or two or

more separate polypeptide chainsmore separate polypeptide chains oror segments of polypeptide chainssegments of polypeptide chains that are that are arranged either antiparallel to each other arranged either antiparallel to each other

(with the (with the N-terminalN-terminal and and C-terminal endsC-terminal ends of the of the -strands-strands alternating alternating (Figure 2.7B),(Figure 2.7B),

oror parallel parallel (with all the N-termini of the (with all the N-termini of the -strands together -strands together (Figure 2.7C).(Figure 2.7C).

When the When the hydrogen bonds are formed hydrogen bonds are formed between the polypeptide backbonesbetween the polypeptide backbones of of separate polypeptide chains, they are separate polypeptide chains, they are termed termed interchain bondsinterchain bonds. .

A A -sheet-sheet can also be can also be formed byformed by a a single polypeptide chainsingle polypeptide chain folding back folding back on itself on itself (Figure 2.7C). (Figure 2.7C). In this case, the In this case, the hydrogen bondshydrogen bonds are are intrachain bondsintrachain bonds..

In In globular proteinsglobular proteins, , -sheets-sheets always always have a right-handed curlhave a right-handed curl, or , or twisttwist, when , when viewed along the polypeptide viewed along the polypeptide backbone. backbone.

[Note: Twisted [Note: Twisted -sheets-sheets often form the often form the core of globular proteins.]core of globular proteins.]

C. C. -Bends (reverse turns)-Bends (reverse turns)

-Bends-Bends reverse the direction of a reverse the direction of a polypeptide chainpolypeptide chain forming a compact, forming a compact, globular shape. globular shape.

They are They are usually found onusually found on the surface of the surface of protein moleculesprotein molecules, and , and often includeoften include charged residuescharged residues. .

[ [ -Bends-Bends were given this name becausewere given this name because they often connect successive strands of they often connect successive strands of antiparallel antiparallel -sheets.]-sheets.]

-Bends-Bends are are generally composed ofgenerally composed of four amino acidsfour amino acids, one of which may be , one of which may be prolineproline — the imino acid that — the imino acid that causes a causes a "kink""kink" in the polypeptide chain. in the polypeptide chain.

GlycineGlycine, the amino acid with the , the amino acid with the smallest R-group, is also frequently smallest R-group, is also frequently found in found in -bends-bends..

- Bends- Bends are are stabilized bystabilized by the formation the formation of of hydrogen and ionic bondshydrogen and ionic bonds..

Lecture 5Lecture 5ContentsContents

- Nonrepetitive secondary structure- Nonrepetitive secondary structure

- Supersecondary structure (Motifs)- Supersecondary structure (Motifs)

Tertiary structure of proteins:Tertiary structure of proteins:– DomainsDomains– Interaction stabilizing tertiary structureInteraction stabilizing tertiary structure– Protein foldingProtein folding– Role of chaperones in protein foldingRole of chaperones in protein folding

Quaternary structure of proteinsQuaternary structure of proteins

D. Nonrepetitive secondary structureD. Nonrepetitive secondary structure

Approximately, Approximately, one half of an average globular one half of an average globular protein is organized into repetitive structuresprotein is organized into repetitive structures, , such as the such as the -helix-helix and/or and/or -sheet-sheet. .

The remainderThe remainder of the polypeptide chain is of the polypeptide chain is described as having a described as having a looploop or or coilcoil conformation. conformation.

These These nonrepetitive secondary structuresnonrepetitive secondary structures are are not "random",not "random", butbut simply have a less regular simply have a less regular structurestructure than those described above. than those described above.

[ [ The termThe term random coilrandom coil refers to therefers to the disordered disordered structure structure obtained when proteins are denaturedobtained when proteins are denatured].].

E. Supersecondary structures (motifs)E. Supersecondary structures (motifs)

Globular proteinsGlobular proteins are constructed byare constructed by combining secondary structural elementscombining secondary structural elements ( (--heliceshelices, , -sheets-sheets, , nonrepetitive sequencesnonrepetitive sequences). ).

These These form primarilyform primarily the the core region core region —that —that is, the interior of the molecule.is, the interior of the molecule.

They are They are connected byconnected by loop regionsloop regions (for (for example, example, -bends at the surface of the -bends at the surface of the protein).protein).

Supersecondary structuresSupersecondary structures are are usually usually produced byproduced by packing side chains from packing side chains from adjacent secondary structural elements adjacent secondary structural elements close to each otherclose to each other. .

-helices-helices and and -sheets-sheets that that are adjacent are adjacent in the amino acid sequencein the amino acid sequence are also are also usually (but not always) usually (but not always) adjacent in the adjacent in the final, folded proteinfinal, folded protein.( Figure 2.8)..( Figure 2.8).

IV. TERTIARY STRUCTURE OF GLOBULAR IV. TERTIARY STRUCTURE OF GLOBULAR PROTEINSPROTEINS

The The primary structureprimary structure of a polypeptide chain of a polypeptide chain determinesdetermines its tertiary structureits tertiary structure. .

[ [ "Tertiary""Tertiary" refers both to therefers both to the folding of folding of domains domains (the basic units of structure and (the basic units of structure and function),function), and the final arrangement of and the final arrangement of domains in the polypeptide.] domains in the polypeptide.]

The The structure of globular proteinsstructure of globular proteins in aqueous in aqueous solutionsolution is is compactcompact, with a , with a high-densityhigh-density (close packing) of the atoms in the core of (close packing) of the atoms in the core of the molecule.the molecule.

Hydrophobic side chainsHydrophobic side chains are are buried in the buried in the interiorinterior, whereas , whereas hydrophilic groupshydrophilic groups are are generally generally found on the surfacefound on the surface of the of the molecule. molecule.

All hydrophilic groupsAll hydrophilic groups (including (including components of the peptide bond) components of the peptide bond) located in located in the interior of the polypeptidethe interior of the polypeptide are are involved involved in hydrogen bonds in hydrogen bonds oror electrostatic electrostatic interactions.interactions.

TheThe -helix -helix and and -sheet structures -sheet structures provide provide maximal hydrogen bondingmaximal hydrogen bonding for peptide bond for peptide bond componentscomponents within the interior of within the interior of polypeptides.polypeptides.

This eliminates the possibility thatThis eliminates the possibility that water water molecules molecules may becomemay become bound to these bound to these hydrophilic groups hydrophilic groups disrupt the integrity of disrupt the integrity of the protein.the protein.

A. DomainsA. Domains

DomainsDomains are are the fundamental functional and the fundamental functional and three-dimensional structural units of a three-dimensional structural units of a polypeptide. polypeptide.

Polypeptide chainsPolypeptide chains that are > 200 amino that are > 200 amino acids in length generally acids in length generally consist of two or consist of two or more domains. more domains.

The The core of a domaincore of a domain is is built from built from combinations of super- secondary structural combinations of super- secondary structural elementselements ( (motifsmotifs).).

Folding of the peptide chainFolding of the peptide chain within a within a domaindomain usually occurs independently usually occurs independently of folding in other domains. of folding in other domains.

Each Each domaindomain has the characteristics of has the characteristics of a small, compact globular proteina small, compact globular protein that that is structurally independent of the other is structurally independent of the other domains in the polypeptide chain.domains in the polypeptide chain.

B. Interactions stabilizing tertiary B. Interactions stabilizing tertiary structurestructure

The unique The unique three-dimensional structurethree-dimensional structure of of each polypeptide is determined by its amino each polypeptide is determined by its amino acid sequence.acid sequence.

Interactions between the amino acid side Interactions between the amino acid side chainschains guide the folding of the polypeptideguide the folding of the polypeptide to to form a compact structure. form a compact structure.

Four types of interactionsFour types of interactions cooperate in cooperate in stabilizing the tertiary structuresstabilizing the tertiary structures of globular of globular proteins.proteins.

11 . .Disulfide bondsDisulfide bonds::

A A disulfide bonddisulfide bond is a “ is a “ covalent linkage formed covalent linkage formed from the sulfhydryl group (- SH) of each of two from the sulfhydryl group (- SH) of each of two cysteine residues, to produce a cystine cysteine residues, to produce a cystine residueresidue” ” (Figure 2.9).(Figure 2.9).

The The two cysteinestwo cysteines may be separated from each may be separated from each otherother by many amino acids in the primary by many amino acids in the primary sequence of a polypeptide, sequence of a polypeptide, oror may even be may even be located on two different polypeptide chainslocated on two different polypeptide chains; ; the folding of the polypeptide chain (s) brings the folding of the polypeptide chain (s) brings the cysteine residues into proximitythe cysteine residues into proximity, , covalent bonding of their side chains.covalent bonding of their side chains.

A A disulfide bonddisulfide bond contributes to the contributes to the stability of the three-dimensional shapestability of the three-dimensional shape of the protein molecule. of the protein molecule.

For exampleFor example, , many disulfide bonds are many disulfide bonds are found in proteinsfound in proteins such as such as immunoglobulinsimmunoglobulins that are secreted by that are secreted by cells. cells.

[ [ TheseThese strong, covalent bondsstrong, covalent bonds stabilize the structure of proteins, stabilize the structure of proteins, and and prevent them from becoming denatured prevent them from becoming denatured in the extracellular environment.]in the extracellular environment.]

22 . .Hydrophobic interactionsHydrophobic interactions::

Amino acids with nonpolar side chainsAmino acids with nonpolar side chains tend to be located in the interior of the tend to be located in the interior of the polypeptide molecule,polypeptide molecule, where they where they associate with other hydrophobic associate with other hydrophobic amino acids. amino acids. (Figure 2.10).(Figure 2.10).

In contrastIn contrast, , amino acids with polar or amino acids with polar or charged side chainscharged side chains tend to be located tend to be located on the surface of the moleculeon the surface of the molecule in in contact with the polar solvent.contact with the polar solvent.

Proteins located in nonpolar (lipid) Proteins located in nonpolar (lipid) environmentsenvironments, , such as a membranesuch as a membrane, , exhibit the exhibit the reverse arrangementreverse arrangement I.e,I.e,

hydrophilic amino acid side chainshydrophilic amino acid side chains are located are located in the interior of the polypeptide, in the interior of the polypeptide, whereaswhereas

hydrophobic amino acidshydrophobic amino acids are located on the are located on the surface of the molecule in contact with the non surface of the molecule in contact with the non polar environment polar environment (Figure 1.4.)(Figure 1.4.)

In each caseIn each case, , the the segregation of R-groupssegregation of R-groups occursoccurs that is energetically most favorable. that is energetically most favorable.

33 . .Hydrogen bondsHydrogen bonds::

Amino acid side chains containing oxygen - or Amino acid side chains containing oxygen - or nitrogen - bound hydrogennitrogen - bound hydrogen, such as , such as in the in the alcohol groups of serine and threoninealcohol groups of serine and threonine, , form form hydrogen bonds with electron-rich atoms, hydrogen bonds with electron-rich atoms, such as the oxygen of a carboxyl group or such as the oxygen of a carboxyl group or carbonyl group of a peptide bond carbonyl group of a peptide bond

(Figure 2.11)(Figure 2.11) & & (Figure 1.6.).(Figure 1.6.).

Formation of hydrogen bondsFormation of hydrogen bonds between polar between polar groups on the surface of proteins and the groups on the surface of proteins and the aqueous solvent aqueous solvent enhances the solubility of enhances the solubility of the protein.the protein.

44 . .Ionic interactionsIonic interactions::

Negatively charged Negatively charged groupsgroups, such as the , such as the carboxyl group carboxyl group (-COO(-COO--)) in the side in the side chain of chain of aspartateaspartate or or glutamateglutamate, can , can interact withinteract with positively positively charged groupscharged groups, such , such as the amino group as the amino group (-NH(-NH33

++ ) ) in the side in the side

chain of chain of lysinelysine (Figure 2.11).(Figure 2.11).

C. Protein foldingC. Protein folding

Interactions between the side chains of Interactions between the side chains of amino acidsamino acids determinedetermine how a long how a long polypeptide chain folds into the polypeptide chain folds into the intricate three-dimensional shapeintricate three-dimensional shape of the of the functional protein. functional protein.

Protein foldingProtein folding, which occurs within the , which occurs within the cell in seconds to minutes, cell in seconds to minutes, employs a employs a shortcut through the maze of all folding shortcut through the maze of all folding possibilities.possibilities.

As a peptide foldsAs a peptide folds, , its amino acid side its amino acid side chainschains are are attracted attracted and and repulsedrepulsed according to their chemical properties. according to their chemical properties.

For exampleFor example, , positivelypositively andand negativelynegatively charged side chainscharged side chains attractattract each othereach other. .

ConverselyConversely, , similarly chargedsimilarly charged side chainsside chains repelrepel each othereach other. .

In additionIn addition, , interactionsinteractions involving involving hydrogen bondshydrogen bonds, , hydrophobic interactionshydrophobic interactions, , and and disulfide bondsdisulfide bonds all all exert an influence exert an influence on the foldingon the folding process. process.

This process This process of of trial and trial and errorerror tests tests many, but not many, but not all, possible all, possible configurationsconfigurations

This results This results in a in a correctly correctly folded proteinfolded protein with a low with a low energy stateenergy state (Figure 2.12).(Figure 2.12).

D. Role of chaperones in protein foldingD. Role of chaperones in protein folding

It is generally accepted that the It is generally accepted that the information needed for correct protein information needed for correct protein foldingfolding is contained inis contained in the primary the primary structure of the polypeptidestructure of the polypeptide. .

It is difficult to explainIt is difficult to explain why most why most proteins when denatured do not proteins when denatured do not resume their native conformationsresume their native conformations under favorable environmental under favorable environmental conditions.conditions.

One answer to this problemOne answer to this problem is that a is that a protein begins to fold in stages during protein begins to fold in stages during its synthesisits synthesis, rather than waiting for , rather than waiting for synthesis of the entire chain to be synthesis of the entire chain to be totally completed. totally completed.

This This limits competing folding limits competing folding configurationsconfigurations made available by made available by longer stretches of nascent peptide.longer stretches of nascent peptide.

In additionIn addition, , a specialized group of a specialized group of proteins, namedproteins, named “ “chaperoneschaperones” ” are are required for the proper foldingrequired for the proper folding of many of many species of proteins. species of proteins.

The The chaperones chaperones “ “heat shock proteinsheat shock proteins” ” interact with the polypeptide at various interact with the polypeptide at various stages during the folding processstages during the folding process..

Some chaperonesSome chaperones are important in are important in keeping the protein unfoldedkeeping the protein unfolded until its until its synthesis is finishedsynthesis is finished, or , or act as act as catalystscatalysts by increasing the rates of the by increasing the rates of the final stages in the folding process. final stages in the folding process.

OthersOthers protect proteinsprotect proteins as they foldas they fold so so that their vulnerable, exposed regions that their vulnerable, exposed regions do not become tangled in unproductive do not become tangled in unproductive encounters.encounters.

V. QUATERNARY STRUCTURE OF V. QUATERNARY STRUCTURE OF PROTEINSPROTEINS

Many proteinsMany proteins consist of a consist of a single single polypeptide chainpolypeptide chain, and are defined as , and are defined as monomeric proteinsmonomeric proteins. .

However, However, othersothers may consist of two or may consist of two or more polypeptide chainsmore polypeptide chains that may be that may be structurally identical or totally unrelated. structurally identical or totally unrelated.

The arrangement of these polypeptide The arrangement of these polypeptide subunits is called the subunits is called the quaternary quaternary structure of the proteinstructure of the protein..

Two subunits, Two subunits, the protein is the protein is ““dimericdimeric”. ”.

Three subunits “Three subunits “trimerictrimeric”. ”.

Several subunits. ‘Several subunits. ‘multimericmultimeric’ ’

Subunits are held together by Subunits are held together by noncovalent interactionsnoncovalent interactions (e.g., (e.g., hydrogen hydrogen bondsbonds, , ionic bondsionic bonds and and hydrophobic hydrophobic interactionsinteractions).).

SubunitsSubunits may either may either

- - function independently of each otherfunction independently of each other, , oror - - work cooperativelywork cooperatively, ,

As in As in hemoglobinhemoglobin, in which the binding , in which the binding of oxygen to one subunit of the tetramer of oxygen to one subunit of the tetramer increases the affinity of the other increases the affinity of the other subunits for oxygen.subunits for oxygen.

Lecture 6Lecture 6

ContentsContents

Denaturation of proteinsDenaturation of proteins

Protein misfolding:Protein misfolding:– AmyloidosisAmyloidosis

– Prion diseasePrion disease

VI. DENATURATION OF PROTEINSVI. DENATURATION OF PROTEINS

Protein denaturationProtein denaturation unfoldingunfolding and and disorganization ofdisorganization of the protein’s secondary and the protein’s secondary and tertiary structurestertiary structures, which are not accompanied , which are not accompanied by hydrolysis of peptide bonds. by hydrolysis of peptide bonds.

Denaturing agentsDenaturing agents include : include :

– heat, heat,

– organic solvents, organic solvents,

– mechanical mixing, mechanical mixing,

– strong acids or bases, strong acids or bases,

– detergents, detergents,

– ions of heavy metals such as lead and ions of heavy metals such as lead and mercury.mercury.

DenaturationDenaturation may, under ideal may, under ideal conditions, be conditions, be reversiblereversible, , the protein the protein refolds into its original native structurerefolds into its original native structure when the denaturing agent is removed. when the denaturing agent is removed.

However, However, most proteinsmost proteins, , once once denatured, remain permanently denatured, remain permanently disordereddisordered. .

Denatured proteinsDenatured proteins are are often insolubleoften insoluble precipitateprecipitate from solution. from solution.

VII. PROTEIN MISFOLDINGVII. PROTEIN MISFOLDING

Protein foldingProtein folding is a is a complexcomplex, , trial trial and error processand error process that can that can sometimes sometimes improperly foldedimproperly folded molecules. molecules.

These These misfolded proteinsmisfolded proteins are are usuallyusually tagged tagged and and degradeddegraded within the cell .within the cell .

However, However, this quality control system is this quality control system is not perfectnot perfect, and , and intracellular or intracellular or extracellular aggregates of misfolded extracellular aggregates of misfolded proteins can accumulateproteins can accumulate, particularly , particularly as individuals' age. as individuals' age.

Deposits of these misfolded proteinsDeposits of these misfolded proteins number of diseases including number of diseases including amyloidosesamyloidoses..

A. AmyloidosesA. Amyloidoses

Misfolding of proteinsMisfolding of proteins may occur may occur spontaneouslyspontaneously, or be , or be caused by a mutation caused by a mutation in a particular gene in a particular gene an altered protein. an altered protein.

In additionIn addition, , some apparently normal some apparently normal proteinsproteins can, after abnormal proteolytic can, after abnormal proteolytic cleavage, cleavage, take on a unique conformational take on a unique conformational statestate formation of formation of longlong, , fibrillar proteinfibrillar protein consisting of consisting of -pleated sheets-pleated sheets..

Accumulation of these spontaneously Accumulation of these spontaneously aggregating proteinsaggregating proteins, “ , “ amyloidsamyloids ” ” many degenerating diseases - many degenerating diseases - particularly in the neurodegenerative particularly in the neurodegenerative disorder, disorder, Alzheimer disease. Alzheimer disease.

The dominant component of the The dominant component of the amyloid plaqueamyloid plaque that accumulates that accumulates inin Alzheimer diseaseAlzheimer disease is is AA, , a peptide of 40 a peptide of 40 to 43 amino acid residuesto 43 amino acid residues..

X-ray crystallographyX-ray crystallography and and infrared infrared spectroscopyspectroscopy demonstratedemonstrate characteristic characteristic -pleated sheet-pleated sheet conformation in conformation in nonbranching fibrils. nonbranching fibrils.

This peptideThis peptide, , when aggregated in a when aggregated in a - - pleated sheet configurationpleated sheet configuration, is , is neurotoxicneurotoxic, and is the central pathogenic , and is the central pathogenic event event cognitive impairmentcognitive impairment characteristic of the disease.characteristic of the disease.

The The AA amyloidamyloid that isthat is deposited in the deposited in the brain in Alzheimer diseasebrain in Alzheimer disease is is derived byderived by proteolytic cleavages from the larger proteolytic cleavages from the larger amyloid precursor proteinamyloid precursor protein - a single - a single transmembrane protein expressed on transmembrane protein expressed on the cell surface in the brain and other the cell surface in the brain and other tissues tissues (Figure 2.13).(Figure 2.13).

The The AA peptides peptides aggregate aggregate generating generating the amyloidthe amyloid that is found in the brain that is found in the brain parenchyma and around blood vessels.parenchyma and around blood vessels.

Most cases of Alzheimer diseaseMost cases of Alzheimer disease are are not genetically basednot genetically based, although at least , although at least 5-10 % of cases are familial5-10 % of cases are familial. .

A second biologic factorA second biologic factor involved in the involved in the development of Alzheimer disease is development of Alzheimer disease is the the accumulation of neurofibillary accumulation of neurofibillary tangles in the braintangles in the brain..

A A key component of these tangled key component of these tangled fibersfibers is is an abnormal form of thean abnormal form of the tau tau proteinprotein, which in its healthy version , which in its healthy version helps in the assembly of the helps in the assembly of the microtubular structure. microtubular structure.

The The defective taudefective tau, however, appears to , however, appears to block the actions of its normal block the actions of its normal counterpart.counterpart.

B. Prion diseaseB. Prion disease

The prion protein (PrP), has been The prion protein (PrP), has been strongly implicated as the causative strongly implicated as the causative agent of agent of transmissible spongiform transmissible spongiform encephalopathiesencephalopathies (TSEs), including; (TSEs), including;– Creutzfeldt-Jakob diseaseCreutzfeldt-Jakob disease in in humanshumans

– ScrapieScrapie in in sheepsheep

– Bovine spongiform encephalopathyBovine spongiform encephalopathy in in cattle cattle ("("mad cow diseasemad cow disease").").

After an extensive series of purification After an extensive series of purification procedures, scientists were astonished procedures, scientists were astonished to find that to find that the infectivity of the agent the infectivity of the agent causing causing scrapiescrapie in sheep was in sheep was associated with a associated with a single protein single protein speciesspecies that was not associated with that was not associated with detectable nucleic acid. detectable nucleic acid.

This infectious proteinThis infectious protein is designated is designated the the prion proteinprion protein..

PrPPrP is is highly resistant to proteolytic highly resistant to proteolytic degradationdegradation, and, , and, when infectiouswhen infectious form form insoluble aggregates of fibrilsinsoluble aggregates of fibrils, , similar to similar to the amyloidthe amyloid found in some other found in some other diseases of the brain. diseases of the brain.

A A noninfectious form of PrPnoninfectious form of PrP, having the , having the same amino acid and gene sequences as same amino acid and gene sequences as the infectious agent, the infectious agent, is present inis present in normal normal mammalian brains on the surface of mammalian brains on the surface of neurons and glial cells. neurons and glial cells.

Thus, Thus, PrPPrP is a is a host proteinhost protein..

No primary structure differencesNo primary structure differences or or alternate posttranslational modifications alternate posttranslational modifications have been have been found between the normal and found between the normal and the infectious forms of the proteinthe infectious forms of the protein. .

The The key tokey to becoming infectiousbecoming infectious apparently apparently lies inlies in changes in the three-dimensional changes in the three-dimensional conformation of PrPconformation of PrP. .

A number of A number of -helices present in -helices present in noninfectious PrPnoninfectious PrP are are replaced byreplaced by -sheets in the infectious form-sheets in the infectious form (Figure 2.14).(Figure 2.14).

Conformational differenceConformational difference confers confers relative relative resistance to proteolytic degradation ofresistance to proteolytic degradation of infectious prionsinfectious prions, and permits them to be , and permits them to be distinguished from the normal PrP in infected distinguished from the normal PrP in infected tissue. tissue.

The infective agentThe infective agent is thus is thus an altered version of an altered version of a normal proteina normal protein, which acts as a , which acts as a "template""template" for for converting the normal protein to the pathogenic converting the normal protein to the pathogenic conformation. conformation.

Transmissible Spongiform EncephalopathiesTransmissible Spongiform Encephalopathies ““TSEsTSEs” are invariably ” are invariably fatalfatal, and , and no treatment is no treatment is currently availablecurrently available that can alter this outcome. that can alter this outcome.

THE ENDTHE END