Secondary, Tertiary, and Quaternary Structure of Proteins PDF

II. SECONDARY, TERTIARY AND QUATERNARY STRUCTURE OF PROTEINS A. Noncovalent Interactions that Stabilize the Higher Levels of Protein Structure 1 structure covalent bonds 2, 3, 4 structures weak, noncovalent interactions  H bonds  hydrophobic interactions  electrostatic bonds  van der Waals forces Hydrogen Bonds are formed whenever Possible component atoms of the peptide backbone tend to form H bonds with one another side chains capable of forming H bonds are usually located on the protein surface form H bonds either with the water solvent or with other surface residues the strengths of H bonds depend to some extent on environment difference in energy between a side chain H bonded to water and that same side chain H bonded to another side chain is usually quite small a H in the protein interior, away from bulk solvent, can provide substantial stabilization energy to the protein although each H bond may contribute an average of only a few kJ per mole in stabilization energy for the protein structure, the number of H bonds formed in the typical protein is very large e.g in -helices, the C=O and N-H groups of every interior residue participate in H bonds Hydrophobic interactions drive Protein folding hydrophobic interactions form because nonpolar side chains of AAs and other nonpolar solutes prefer to cluster in a nonpolar environment rather than to intercalate in a polar solvent such as water forming of hydrophobic “bonds” minimizes the interaction of nonpolar residues with water such clustering is entropically driven and is the principal impetus for protein folding side chains of the AAs in the interior or core of the protein structure are almost exclusively hydrophobic polar AAs are much less common in the interior of a protein protein surface may consist of both polar and nonpolar residues Ionic interactions usually occur on the Protein surface ionic interactions arise either as electrostatic attractions between opposite charges or repulsions between like charges AA chains carry (+) charges or (-) charges N-terminal and C-terminal residues of a protein or peptide chain usually exist in ionized states and carry (+) or (-) charges charged residues are normally located on the protein surface, where they may interact optimally with the water solvent ionic interactions between charged groups on a protein surface are often complicated by the presence of salts in the solution e.g. the ability of a K to attract a nearby E may be weakened by dissolved salts the Na+ and Cl- ions are highly mobile, compact units of charge, compared to the AA side chains, and thus compete effectively for charged sites on the protein ionic interactions among AA residues on protein surfaces may be damped out by high concentrations of salts nevertheless, these interactions are important for protein stability Van der Waals interactions are ubiquitous both attractive forces and repulsive forces are included in van der Waals interactions attractive forces are due primarily to instantaneous dipole-induced dipole interactions that arise because of ﬂuctuations in the e- charge distributions of adjacent nonbonded atoms individual van der Waals interactions are weak ones but many such interactions occur in a typical protein by sheer force of numbers, they can represent a signiﬁcant contribution to the stability of a protein individual van der Waals interactions are weak ones (stabilization energies of 0.4 to 4.0 kJ/mol) many such interactions occur in a typical protein, and by sheer force of numbers, they can represent a signiﬁcant contribution to the stability of a protein II. SECONDARY, TERTIARY AND QUATERNARY STRUCTURE OF PROTEINS B. Elements of Secondary Structure and their Formation The -helix is a key 2 Structure 2 structure local conformations of the polypeptide that are stabilized by H bonds the H bonds that make up 2 structures involve the amide proton of one peptide group and the carbonyl oxygen of another A H bond between the backbone C=O of Ala191 and the backbone N- H of Ser147 in the acetylcholine- binding protein of a snail these structures tend to form in cooperative fashion and involve substantial portions of the peptide chain when a number of H bonds form between portions of the peptide chain in this manner, 2 basic types of structures result  -helices  -pleated sheets -helix 1 turn of the helix = 3.6 AA residues each AA residue extends 1.5 Å (0.15 nm) along the helix axis pitch of the helix = 5.4 Å (0.54 nm) (3.6 x 1.5 Å) helix diameter = 6 Å each peptide carbonyl is H bonded to the peptide N-H group four residues farther up the chain all of the H bonds lie parallel to the helix axis all of the carbonyl groups are pointing in one direction along the helix axis the N-H groups are pointing in the opposite direction the number of residues involved in a given -helix varies from helix to helix and from protein to protein average = 10 residues per helix all of the H bonds point in the same direction along the -helix axis with one exception, all of the twenty common AAs are found frequently in α-helices each of the peptide bond possesses a dipole moment that arises from the polarities of the N-H and C=O groups the helix has a substantial dipole moment, with a + at the N-terminus and a - at the C- terminus (-)ly charged ligands (phosphates) frequently bind to proteins near the N- terminus of an -helix (+)ly charged ligands are only rarely found to bind near the C- in a typical -helix of 12 (or n) residues, there are 8 (or n  4) H bonds the ﬁrst 4 amide hydrogens and the last 4 carbonyl oxygens cannot participate in helix H bonds nonpolar residues situated near the helix termini can be exposed The -Pleated Sheet is a Core Structure in Proteins -pleated sheet can be visualized by laying thin, pleated strips of paper side by side to make a “pleated sheet” of paper each strip of paper can be pictured as a single peptide strand in which the peptide backbone makes a zigzag pattern along the strip, with the -Cs lying at the folds of the pleats parallel -pleated sheet adjacent chains run in the same direction distance between residues is 0.325 nm the H bonds formed are bent signiﬁcantly parallel -pleated sheet typically large structures those composed of fewer than 5 strands are rare distribute hydrophobic side chains on both sides of the sheet antiparallel -pleated sheet adjacent chains run in opposite directions distance between residues is 0.347 nm antiparallel -pleated sheet may consist of as few as 2 strands usually arranged with all their hydrophobic residues on one side of the sheet requires an alternation of hydrophilic and hydrophobic residues in the primary structure of peptides because every other side chain projects to the same the arrangement of successive amide planes in a β-sheet has a pleated appearance due to the tetrahedral nature of the Cα atom the H bonds in this structure are interstrand rather than intrastrand optimum formation of H bonds in the parallel pleated sheet results in a slightly less extended conformation than in the antiparallel sheet the side chains in the pleated sheet are oriented perpendicular or normal to the plane of the sheet, extending out from the plane on alternating sides parallel β-sheets tend to be more regular than antiparallel β- sheets Helix–Sheet Composites Provide Flexibility and Strength in Spider Silk although the intricate designs of spider webs are eye (and ﬂy) catching, the composition of web silk itself is even more remarkable spider silk, a form of keratin, is synthesized in special glands in the spider’s abdomen the silk strands produced by these glands are both strong and Dragline silk (that from which the spider hangs) has a tensile strength of 200,000 psi, — stronger than steel and similar to Kevlar this same silk ﬁber is also ﬂexible enough to withstand strong winds and other natural stresses this combination of strength and ﬂexibility derives from the Spider web silks as keratin protein is extruded from the spider’s glands, it endures shearing forces that break the H bonds stabilizing keratin a-helices these regions then form microcrystalline arrays of β- sheets these microcrystals are surrounded by the keratin strands, which adopt a highly disordered the β-sheet microcrystals contribute strength, and the disordered array of helix and coil make the silk strand ﬂexible the resulting silk strand resembles modern human- engineered composite materials and products (ex. tennis racquets) -Turns Allow the Protein Strand to Change Direction polypeptide chains must possess the capacity to bend, turn, and reorient themselves to produce compact, globular structures -turn (tight turn or -bend) peptide chain forms a tight loop with the carbonyl oxygen of one residue hydrogen bonded with the amide proton of the residue 3 positions down the chain H bond makes the -turn a relatively stable structure allows the protein to reverse the direction of its peptide chain 2 major types of -turns: Type I & Type II G is sterically the most adaptable of the AAs, and it accommodates conveniently to other steric Pro has a cyclic structure and a ﬁxed angle, so it forces the formation of a -turn facilitates the turning of a polypeptide chain upon itself promote formation of antiparallel  -pleated sheets type I turns are more common than type II Pro ﬁts best in the 3 and 2 position of the type I and type II turns, respectively gly or small polar residues ﬁt best in the 3 position of type II turn II. SECONDARY, TERTIARY AND QUATERNARY STRUCTURE OF PROTEINS C. Folding of Polypeptides into Three-Dimensional Protein Structure 3 structure arrangement of all atoms of a single polypeptide chain in 3-D space all of the info needed to fold the protein into its native 3 structure is contained within the 1 structure of the peptide chain itself 1st determinations of the 3 structure of a protein (late 1950s) John Kendrew: Mb Max Perutz: Hb Important principles 1. 2 structures form whenever possible as a consequence of the formation of large numbers of H bonds 2. -helices and -sheets often associate and pack close together in the protein  no protein is stable as a single- layer structure 3. because the peptide segments between 2 structures in the protein tend to be short and direct, the peptide does not execute complicated twists and knots as it moves from one region of 2 structure to another 4. proteins generally fold so as to form the most stable structures possible Stability of most proteins arises from 1. formation of large numbers of intramolecular H bonds 2. reduction in the surface area accessible to solvent that occurs upon folding 2 factors that lie at the heart of these 4 principles 1. proteins are typically a mixture of hydrophilic and hydrophobic AAs  imagine a protein composed only of polar and charged AAs  every side chain could H bond to water  protein will not form a compact, folded structure 2. consider a protein composed of a mixture of hydrophilic and hydrophobic residues  the hydrophobic side chains cannot form H bonds with water  their presence will disrupt the H- bonding structure of water itself  to minimize this, the hydrophobic groups will tend to cluster together  hydrophobic effect induces formation of a compact structurethe folded protein a potential problem with this rather simple folding model is that polar backbone N-H and C=O groups on the hydrophobic residues accompany the hydrophobic side chains into the folded protein interior this would be energetically costly to the protein Fibrous Proteins usually play a Structural Role 3 large classes of proteins based on their structure and solubility ﬁbrous proteins globular proteins membrane proteins ﬁbrous proteins contain polypeptide chains organized approximately parallel along a single axis, producing long ﬁbers or large sheets tend to be mechanically strong and resistant to solubilization in water and dilute salt solutions play a structural role in nature -keratins predominant constituents of claws, ﬁngernails, hair and horns in mammals the structure is dominated by - helical segments of polypeptide Both type I and type II a-keratin molecules have sequences consisting of long, central rod domains with terminal cap domains. Asterisks denote domains of variable length. Steinert, P., and Parry, D., 1985. Intermediate ﬁlaments: Conformity and diversity of expression and structure. Annual Review of Cell Biology 1:41–65; and Cohlberg, J., 1993. The rod domains form coiled coils consisting of left-twisted right-handed a-helices. These coiled coils form protoﬁlaments that then wind around each other in a right- handed twist. Keratin ﬁlaments consist of twisted protoﬁbrils (each a bundle of four coiled coils). Steinert, P., and Parry, D., 1985. Intermediate ﬁlaments: Conformity and diversity of expression and structure. Annual Review of Cell Biology 1:41–65; and Cohlberg, Steinert, P., and Parry, D., 1985. Intermediate ﬁlaments: Conformity and diversity of expression and structure. Annual Review of Cell Biology 1:41–65; and Cohlberg, J., 1993. in other forms of keratin, covalent disulﬁde bonds form between Cys residues of adjacent molecules, making the overall structure even more rigid, inextensible, and insoluble how and where these disulﬁdes form determines the amount of curling in hair and wool ﬁbers when a hairstylist creates a permanent wave (perm) in a hair salon, disulﬁdes in the hair are ﬁrst reduced and cleaved, then reorganized and reoxidized to change the degree of curl or wave a “set” created by wetting the hair, setting it with curlers, and then drying it represents merely a rearrangement of the H bonds between helices and between ﬁbers on humid or rainy days, the H bonds in curled hair may rearrange, and the hair becomes frizzy Fibroin and -Keratin: -Sheet Proteins ﬁbroin proteins found in silk ﬁbers in the cocoons of the silkworm,Bombyx mori, and also in spiderwebs stacked antiparallel -sheets -keratins found in bird feathers stacked -sheets Collagen: a Triple Helix Collagen a rigid, inextensible ﬁbrous protein principal constituent of connective tissue in animals, including tendons, cartilage, bones, teeth, skin, and blood vessels the high tensile strength of collagen ﬁbers makes possible the various animal activities: running and jumping that put severe stresses on joints and skeleton broken bones and tendon and cartilage injuries to knees, elbows, and other joints involve tears or hyperextensions of the collagen matrix in these tissues tropocollagen basic structural unit of collagen MW = 285,000 consists of 3 intertwined polypeptide chains, each about 1000 AAs in length about 300 nm long about 1.4 nm in diameter Kinds of collagen Type I collagen most common 2 identical peptide chains: 1(I), 2(I) predominates in bones, tendons & skin Type II collagen found in cartilage Type III collagen found in blood vessels collagen has an AA composition that is unique and is crucial to its 3- D structure and its characteristic physical properties nearly 1 residue out of 3 is a Gly and the Pro content is unusually high 3 unusual modiﬁed AAs in collagen  4-hydroxyproline (Hyp)  3-hydroxyproline  5-hydroxylysine (Hyl) proline and Hyp together compose up to 30% of the residues of collagen because of the high content of Gly, Pro, and Hyp, collagen ﬁbers are incapable of forming -helices and -sheets collagen polypeptides intertwine to form a unique right-handed triple helix, with each of the 3 strands arranged in a left-handed helical fashion collagen helix is much more extended, with a rise per residue along the triple helix axis of 2.9 Å 3.3 residues per turn long stretches of the polypeptide sequence are repeats of a Gly-x-y motif, x = Pro; y = Pro or Hyp every 3rd residue is a Gly triple helix structure is further stabilized and strengthened by the formation of interchain H bonds involving Hyp Globular Proteins Mediate Cellular Function globular proteins are far more numerous than ﬁbrous proteins the functional diversity and versatility derive from  the large number of folded structures that polypeptide chains can adopt  varied chemistry of the side Helices and Sheets make up the Core of most Globular Proteins bovine ribonuclease A a small protein (12.6 kD, 124 residues) a few short -helices, a broad section of antiparallel -sheet, a few -turns, and several peptide segments without deﬁned 2 structure the space between the helices and sheets in the protein interior is ﬁlled eﬃciently and tightly with mostly hydrophobic AA side chains most polar side chains in ribonuclease face the outside of the protein structure and interact with solvent water the folding of a globular protein could be viewed as the aggregation of multiple elements of 2 structure most peptide segments that form helices, sheets, or β turns in proteins are mostly disordered in small model peptides that contain those AA sequences hydrophobic interactions and other noncovalent interactions with the rest of the protein must stabilize these relatively unstable helices, sheets, and turns in the folded protein the cores of most globular and membrane proteins consist almost entirely of -helices and -sheets the highly polar N-H and C=O moieties of the peptide backbone must be neutralized in the hydrophobic core of the protein the extensively H-bonded nature of -helices and -sheets is ideal for this purpose these structures effectively stabilize the polar groups of the peptide backbone in the protein core the framework of sheets and helices in the interior of a globular protein is constant and conserved in both sequence and structure these structures effectively stabilize the polar groups of the H bonds in the nonpolar core of globular proteins are typically 5–6 kJ/mole stronger than those on the protein surface whereas the interior of a globular protein is composed of conserved sheets and helices, the surface of a globular protein is different in several ways much of the protein surface is composed of the loops and tight turns that connect the helices and sheets of the protein core, although helices and sheets may also be found on the surface the result is that the surface of a globular protein is often a complex landscape of different structural elements coil or random coil the segments of the protein that are neither helix, sheet, nor turn most of these loop segments are neither coiled nor random these structures are every bit as organized and stable as the deﬁned secondary structures they just don’t conform to any frequently recurring pattern Water Molecules on the Protein Surface Stabilize the Structure a globular protein’s surface structure is affected by the surrounding water molecules many of the polar backbone and side chain groups on the surface of a globular protein make H bonds with solvent water molecules there are often several such water molecules per AA residue, and some are in ﬁxed positions Actinidin an enzyme from kiwi fruit that cleaves polypeptide chains at Arg in some globular proteins, it is common for one face of an - helix to be exposed to the water solvent, with the other face toward the hydrophobic interior of the protein the outward face of such an amphiphilic helix consists mainly of polar and charged residues, whereas the inward face contains a surface helix, residues 153 to 166 of ﬂavodoxin from Anabaena the helical wheel presentation of this helix readily shows that one face contains 4 hydrophobic residues and that the other is almost entirely Packing Considerations the 2 and 3 structures of ribonuclease A and other globular proteins illustrate the importance of packing in 3 structures 2 structures pack closely to one another and also intercalate with extended polypeptide chains if the sum of the van der Waals volumes of a protein’s constituent AAs is divided by the volume occupied by the protein, packing densities of 0.72 to 0.77 are typically obtained these packing densities are similar to those of a collection of solid spheres even with close packing, approximately 25% of the total nearly all of this space is in the form of very small cavities cavities the size of water molecules or larger do occasionally occur they make up only a small fraction of the total protein volume provide ﬂexibility for proteins facilitate conformation changes and a wide range of protein dynamics Protein Domains are Nature’s Modular Strategy for Protein Design proteins composed of about 250 AAs or less have a simple, compact globular shape larger proteins are usually made up of 2 or more recognizable and distinct structures domains or modules compact, folded protein structures that are usually stable by themselves in aqueous solution N-terminal domain (right) DNA (middle) C-terminal domain (left) Ton-EBP, a two domain DNA-binding protein, joined by a short segment of the peptide chain most domains consist of a single continuous portion of the protein sequence, but in some proteins the domain sequence is interrupted by a sequence belonging to some other part of the protein that may even form a separate domain malonyl CoA:ACP transacylase a metabolic enzyme consisting of 2 subdomains large subdomain (blue), residues 1–132 and 198–316  consists of a β-sheet surrounded by 12 α-helices small subdomain (gold), residues 133–197  consists of a 4-stranded antiparallel β-sheet and two α-helices it is likely that proteins consisting of multiple domains (multiple functions) evolved by the fusion of genes that once coded for separate proteins this would require gene duplication to be common in nature analysis of completed genomes has conﬁrmed that approximately 90% of domains in eukaryotes have been duplicated the protein domain is a fundamental unit in evolution many proteins have been assembled by duplicating domains and then combining them in different ways many proteins are assemblies constructed from several individual domains, and some proteins contain multiple copies of the same domain The tertiary structures of 9 domains that are frequently duplicated Several proteins that contain multiple copies of one or more of Denaturation Leads to Loss of Protein Structure and Function denaturation loss of protein structure and function denaturation of the protein ovalbumin during the cooking of an egg about 10% of the mass of an egg white is protein 54% ovalbumin when a chicken egg is cracked open, the “egg white” is a nearly transparent, viscous ﬂuid cooking turns this ﬂuid to a solid, white mass the egg white proteins have unfolded and have precipitated out of solution the unfolded proteins have aggregated into a solid mass as a protein solution is heated slowly, the protein remains in its native state until it approaches a characteristic melting temperature, Tm as the solution is heated further, the protein denatures over a narrow range of temperatures aroundTm denaturation over a very small temperature range is evidence of a two-state transition between the native and the unfolded states of the protein this implies that unfolding is an all-or-none process when weak forces are disrupted in one part of the protein, the entire structure breaks down Denaturation of ribonuclease ribonuclease A (blue) ribonuclease B (red) lose activity above 55° C most proteins can be denatured below the transition temperature by a variety of chemical agents  acid or base  organic solvents  detergents  denaturing solutes  guanidine hydrochloride  urea denaturation in all these cases involves disruption of the weak forces that stabilize proteins covalent bonds are not affected acids and bases cause protonation and deprotonation of dissociable groups on the protein, altering ionic interactions and H bonds organic solvents and detergents disrupt hydrophobic interactions that bury nonpolar groups in the protein interior guanidine hydrochloride and urea direct effects (binding to hydrophilic groups on the protein) indirect effects (altering the structure and dynamics of the water solvent good H-bond donors and acceptors Denaturation of chymotrypsin in guanidine-HCl Anﬁnsen’s Classic Experiment proved that Sequence determines Structure all the info needed to fold a polypeptide into its native structure is contained in the AA sequence 1950s, Christian Anﬁnsen and his coworkers at the National Institute of Health denaturation and renaturation studies of ribonuclease A ribonuclease A from bovine pancreas 124 residues 4 disulﬁde bonds ribonuclease cleaves chains of ribonucleic acid only ribonuclease in its native structure possesses enzyme activity treated solutions of ribonuclease with a combination of urea and mercaptoethanol urea unfolded the protein mercaptoethanol reduced the disulﬁde bridges all enzymatic activity of ribonuclease was destroyed removing the mercaptoethanol restored only 1% of the enzyme activity formation of random disulﬁde bridges with 8 Cys residues, there are 105 possible ways to make 4 disulﬁde bridges (7 x 5 x 3 x 1 = 105) removing mercaptoethanol and urea at the same time: the polypeptide was able to fold into its native structure, the correct set of 4 disulﬁdes reformed, and full enzyme activity was recovered this experiment demonstrated that the info needed for protein folding resided entirely within the AA sequence of the protein itself Anﬁnsen shared the 1972 Nobel Prize in Chemistry with William H. Stein and Stanford Moore Is there a Single Mechanism for Protein Folding? Christian Anﬁnsen’s experiments demonstrated that proteins can fold reversibly native structures of at least some globular proteins are thermodynamically stable states Cyrus Levinthal (1968) so many conformations are possible for a typical protein that the protein does not have suﬃcient time to reach its most stable conformational state by sampling all the possible conformations Levinthal’s paradox consider a protein of 100 AAs assume that there are only 2 conformational possibilities per AA 2100 = 1.27 x 1030 possibilities allow 10-13 s for the protein to test each conformational possibility in search of the overall energy minimum: Levinthal’s paradox led protein chemists to hypothesize that proteins must fold by speciﬁc folding pathways Several consistent themes have emerged from these studies 1. 2 structures probably form ﬁrst 2. nonpolar residues may aggregate or coalesce hydrophobic collapse 3. subsequent steps probably involve formation of long-range interactions between 2 structures or involving other hydrophobic interactions 4. the folding process may involve one or more intermediate states, including transition states and molten globules even in the denatured state, many proteins possess small amounts of residual structure due to hydrophobic interactions, with strong interresidue contacts between side chains that are distant in the native protein structure such interactions, together with small amounts of 2 structure, may act as sites of nucleation for the folding process the molten globule is a ﬂexible but compact form characterized by signiﬁcant amounts of 2 structure, no precise 3 structure, and a loosely packed hydrophobic core the process of folding is complex sophisticated simulations have provided models of folding (unfolding) pathways for many proteins multiple folding pathways Computer simulations of folding and unfolding of proteins chymotrypsin inhibitor 2 (CI2) and barnase D = denatured, I = intermediate, TS = transition state, P = physiological Daggett, V., and Fersht, A. R., 2003. Is there a unifying mechanism for protein folding? Ken Dill (University of California, San Francisco) the folding process can be pictured as a funnel of free energies—an energy landscape the rim at the top of the funnel represents the many possible unfolded states for a polypeptide chain characterized by high free energy signiﬁcant conformational entropy polypeptides fall down the wall of the funnel as contacts made between residues establish different folding possibilities the narrowing of the funnel reﬂects the smaller number of available states as the protein approaches its ﬁnal state bumps or pockets on the funnel walls represent partially stable intermediates in the folding pathway the most stable (native) folded state of the protein lies at the bottom of the funnel Motion in Globular Proteins proteins are dynamic structures most globular proteins oscillate and ﬂuctuate continuously about their average or equilibrium structures ﬂexibility is essential for a variety of protein functions: ligand binding, E catalysis, and E regulation motions of proteins may be motions of individual atoms, groups of atoms, or even whole sections of the protein may arise either from thermal energy or from speciﬁc, triggered conformational changes in the protein 1. atomic ﬂuctuations e.g. vibrations random very fast usually occur over small distances arise from the kinetic energy within the protein a function of temp in the tightly packed interior of the typical protein, atomic movements of 1  Å are typical the closer to the surface of the protein, the more movement can occur on the surface atomic movements of several Å are possible 2. collective motions slower motions may extend over larger distances movements of a group of atoms covalently linked in such a way that the group moves as a unit range from a few atoms to hundreds of atoms 2 types of collective motions 1. those that occur quickly but infrequently (tyr ring ﬂips) those that occur slowly (hinge- bending movement between protein domains) e.g. the two antigen-binding domains of immunoglobulins move as relatively rigid units to selectively bind separate antigen molecules also arise from thermal energies in the protein and operate on a timescale of 10-12 to 10-3 s a tyr ring ﬂip takes only a ps but such ﬂips occur only about once every ms 3. conformational changes motions of groups of atoms (individual side chains) or even whole sections of proteins occur on a time scale of 10-9 to 103 s distances covered can be as large as 1 nm occur in response to speciﬁc stimuli arise from speciﬁc interactions may occur in response to speciﬁc stimuli arise from speciﬁc interactions within the protein the cis-trans isomerization of pro residues in proteins occurs over an even longer time scale—typically 101 to 104 s conversion of even a single pro from its cis to its trans conﬁguration can alter a protein proline cis-trans isomerizations sometimes act as switches to activate a protein or open a channel across a membrane The Folding Tendencies and Patterns of Globular Proteins globular proteins adopt the most stable 3 structure possible the polypeptide itself does not usually form simple straight chains an extended peptide chain, being composed of L-AAs, has a tendency to twist slightly in a right-handed direction this tendency is apparently the basis for the formation of a variety of 3 structures having a right- handed sense right-handed twists in -sheets and right-handed crossovers in parallel –sheets right-handed twisted -sheets are found at the center of a number of proteins and provide an extended, highly stable structural core 2 types of connections between -strands 1. hairpins connect adjacent antiparallel - strands 2. crossovers connect adjacent (or nearly adjacent) parallel -strands tendency of an extended polypeptide chain to adopt a right- handed twist structure protein domains with different numbers of layers of backbone structure 2 layers of -helix a -sheet layer between two layers of helix, 3 layers The concentric “layers” of - sheet (inside) and -helix (outside). Hydrophobic residues are buried between these concentric layers in the same a -sheet layer sandwiched manner as in the planar layers between 4 layers of -helix, 5- of the other proteins. layer structure Hydrophobic layers are shaded Proteins can be Classiﬁed on the Basis of Folding Patterns the Structural Classiﬁcation of Proteins database (SCOP2) uses both automated algorithms and manual inspection to describe the structural and evolutionary relationships between all the proteins whose structures are known 4 properties of all proteins in SCOP2 protein types structural classes folding relationships evolutionary relationships 4 groups under protein type soluble ﬁbrous membrane intrinsically disordered proteins 4 broad groups of protein structural classes all  proteins all  proteins / proteins (intermingled)  +  proteins (separated) Folding relationships families superfamilies folds family closely related proteins that show clear evidence of evolutionary origin superfamily brings together more distantly related protein domains, again based on common evolutionary origin fold number, arrangement, and connections of 2 structure elements although the numbers of unique folds ( 1500), superfamilies ( 2600), and families ( 5600) increase as more genomes are known and analyzed, the number of protein domains in nature is large but limited How many proteins can we expect to identify and understand someday? the Protein Data Bank curates more than 200,000 structures at present approximately 103 to 105 genes per organism and approximately 14 million species of living organisms on earth when sequence and structure are both conserved, the evolutionary relationship is stronger structure depends on sequence, function depends on structure it is tempting to imagine that all proteins of a similar structure should share a common function, but this is not always true TIM barrel triose phosphate isomerase a common protein fold consisting of 8 α-helices and 8 β- strands that alternate along the peptide backbone to form a doughnut–like 3 structure an enzyme that interconverts ketone and aldehyde substrates in the breakdown of sugars other TIM barrel proteins carry out very different functions reduction of aldose sugars hydrolysis of phosphate esters not all proteins of similar function possess similar domains catalyze the same reaction, but they bear little structural similarity to each other Molecular Chaperones are Proteins that help other Proteins to Fold molecular chaperones are essential for correct folding of certain polypeptide chains in vivo their assembly into oligomer preventing inappropriate liaisons with other proteins during their synthesis, folding, and transport heat shock proteins (Hsp) induced in cells by elevated temperature or other stress Hsp70 a 70-kD heat shock protein chaperonins (Cpn60s or Hsp60s) a class of 60-kD heat shock proteins GroEL anE. coli protein shown to affect the folding of several proteins Some Proteins are Intrinsically Unstructured intrinsically unstructured proteins (IUPs) or intrinsically disordered proteins (IDPs) exist and function normally in a partially unfolded state do not possess uniform structural properties but are essential for basic cellular functions Instrinsically Unstructured Proteins an almost complete lack of folded structure and an extended conformation with high intramolecular ﬂexibility essential for many basic cellular functions  protein solubility enhancement  regulation of protein lifetimes via control of proteolysis  control of protein–protein interactions a unique combination of high net charge and low overall hydrophobicity higher levels of E, K, R, G, Q, S, P low amounts of I, L, V, W, F, Y, C, N predictive analysis of whole genomes indicates that 2% of archaeal and 4.2% of bacterial proteins probably contain long regions of disorder 25% to 30% of eukaryotic proteins are mostly disordered, and more than half of eukaryotic proteins have long regions of disorder the human proteome is estimated to have between 35% and 50% disordered residues Prevalence of disordered segments in proteins may reﬂect 2 different cellular needs 1. disordered proteins are more malleable and thus can adapt their structures to bind to multiple ligands, including other proteins each such interaction could provide a different function in the cell 2. compared with compact, folded proteins, disordered segments in proteins appear to be able to form larger intermolecular interfaces to which ligands, such as other proteins, could bind folded proteins might have to be 2 to 3 times larger to produce the binding surface possible with a disordered protein larger proteins would increase cellular crowding or could increase cell size by 15% to 30% the ﬂexibility of disordered proteins may reduce protein, genome, and cell sizes Artiﬁcial Intelligence Algorithms Predict Protein Structures Accurately AlphaFold an artiﬁcial intelligence program developed by Alphabet/Google’s DeepMind subsidiary that can predict 3-dimensional structures of proteins with high accuracy millions of structures of proteins from the human proteome and other key proteins of interest have been generated, based only on the primary structures of these proteins AlphaFold2 software open-source AlphaFold Protein Structure Database provides open access to protein How do Protein Subunits interact at the 4 Level of Protein Structure many proteins exist in nature as oligomers oligomers complexes composed of noncovalent assemblies of 2 or more monomer subunits liver alcohol dehydrogenase homodimer composed of identical subunits within each subunit is a 6-stranded parallel sheet between the 2 subunits is a 2-stranded antiparallel sheet alcohol dehydrogenase oxidizes alcohol consumed in a beer or mixed drink in the liver glycogen phosphorylase a homodimeric enzyme which when controlled by hormonal signals modulate blood sugar levels Hb carries oxygen in the blood contains 2 each of 2 different subunits quaternary structure the way in which separate folded monomeric protein subunits associate to form the oligomeric protein There is Symmetry in 4 Structure cyclic symmetry dihedral symmetry cyclic symmetry the subunits are arranged around a single rotation axis if there are 2 subunits, the axis is referred to as a twofold rotation axis rotating the 4 structure 180° about this axis gives a structure identical to the original one with 3 subunits arranged about a threefold rotation axis, a rotation of 120° about that axis gives an identical structure Open 4 Structures can Polymerize all of the 4 structures considered have been closed structures, with a limited capacity to associate closed structures additional subunits cannot be bound many proteins in nature associate to form open heterologous structures open structures can polymerize more or less indeﬁnitely, creating structures that are both esthetically attractive and functionally important to the cells or tissue in which they exist tubulin αβ-dimeric protein that polymerizes into long, tubular structures that are the structural basis of cilia, ﬂagella, and the cytoskeletal matrix the microtubule thus formed consist of 13 parallel ﬁlaments arising from end-to-end aggregation of the tubulin dimers Human immunodeﬁciency virus (HIV) causative agent of AIDS enveloped by a spherical shell composed of hundreds of coat protein subunits, a large-scale, but closed, 4 association Structural and Functional Advantages to 4 Association Important consequences when protein subunits associate in oligomeric structures 1. stability reduction of the protein’s surface-to-volume ratio surface-to-volume ratio becomes smaller as the radius of any particle or object decreased surface-to-volume ratios usually result in more stable proteins subunit association may also serve to shield hydrophobic residues from solvent water 2. genetic economy and eﬃciency oligomeric association of protein monomers is genetically economical for an organism less DNA is required to code for a monomer that assembles into a homomultimer than for a large polypeptide of the same molecular mass all of the information that determines oligomer assembly and subunit–subunit interaction is contained in the genetic material needed to code for the monomer e.g. HIV protease, a dimer of identical subunits, performs a catalytic function similar to homologous cellular enzymes that are single polypeptide chains of 3. bringing catalytic sites together many enzymes derive at least some of their catalytic power from oligomeric associations of monomer subunits the monomer may not constitute a complete enzyme active site formation of the oligomer may bring all the necessary catalytic groups together to form an active enzyme e.g. the active sites of bacterial glutamine synthetase are formed from pairs of adjacent subunits the dissociated monomers are inactive 4. cooperativity most oligomeric enzymes regulate catalytic activity by means of subunit interactions, which may give rise to cooperative phenomena multisubunit proteins typically possess multiple binding sites for a given ligand if the binding of ligand at one site changes the aﬃnity of the protein for ligand at the other binding sites, the binding is said to be cooperative allostery information transfer in this manner across long distances in proteins “at another site” positive cooperativity increases in aﬃnity at subsequent sites negative cooperativity decreases in aﬃnity at subsequent sites binding of ligand to one subunit can inﬂuence the binding behavior at the other subunits

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