Protein Biochemistry CHEM 660
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Structure and Function of Fibrous Proteins Chapter I4 Introduction to Protein Structure Fibrous Proteins Most fibrous proteins play structural roles and have regular extended structures that represent a level of organization intermediate between pure secondary structure and tertiary structures of globular proteins Fibrous proteins are highly elongated moleculesassemblies dominant structural motif proteins In fibrous proteins the secondary structure becomes the Fibrous protein are structurally simple relative to globular Information regarding the structures and interactions involved in fibrous proteins are limited due to their limited solubility in water and the fact that they do not readily form crystals for Xray diffraction Keratins 0 Found in hair wool nails claws quills hooves the outer layer of skin Keratin is mechanically durable and chemically unreactive 0 Classi ed as 0 keratins mammals or B keratins birds and reptiles 0 May comprise as much as 85 of cellular protein Keratins are the most abundant proteins in epithelial cells 0 Keratin a Helix of Helices 0 A wide variety of the structural proteins involved in maintaining cell shape organizing cytoplasm and movement are coiled coils of two or three 0helices wound around each other forming a lefthanded superhelix ln forming a coiledcoil structure the 0 helices are distorted slightly from their normal geometry In forming a coiledcoil structure 0helices come together and interact with each other through hydrophobic residues that form apolar stripes along one face of each helix Distortion of the 0helices results in a slightly tighter winding of the helix 35 residues per turn Therefore ahelices which form coiled coils are characterized by a regularity of the amino acid sequence which repeats every seven residues heptad repeat a b c d e fg l t g E g 2 D D gt g 3 L MMN 0 Keratin a Helix of Helices Heptad repeat abcdefg amino acid residues with hydrophobic side chains in positions a and 0 position is usually Leu lle orAla d position is usually Leu orAla Inter helix electrostatic interactions generally occur between residues in the e and g positions eg and e g Usually see charged and polar amino acids with interact favorably with water in remaining positions 6 1quot 7quot ln coiledcoil proteins with two chains the helices are generally align in parallel and in exact register which is believed to maximize stability of the coiledcoil structure lm 1 AAA Coiled coils can be homo or hetero in nature 0 lu de v Isolated 0helices are not terribly stable but are very stable in coiled coils due to the many stabilizing interactions between the helices 0 Keratin a Helix of Helices Higher order construction of O keratin is poorly understoo Struaure Of 0 keratin is dominated by a a Dimer blProtulilament it Micmlihril coiledcoil extending 300330 residues on average The coiled coil is flanked by N and Cterminal domains Nterminal heads Coiled and Cterminal domains vary greatly in coil rod N size l0 500 residues and they show greater sequence variability than the coiled coil regions Cterminal Terminal domains may contribute to tails functional speci city Keratins are often classi ed as intermediate laments IF components of the large group of cytoskeletal lamentous proteins At least six different lF have been identi ed Types l and II identi ed as acidic and basic keratins based on the nature of the N and Cterminal domains of the keratin coiledcoils O Keratin a Helix of Helices 39 Acidic and basic monomers come together to form a heterodimeric coiledcoil Higher order conStrUCtlon 0f 0 keratin is a Dimer blProtofilament c Micro hril poorly understood Mammal heads Q 39 Twou protofilaments come together to a a 50A protofibril coiledcoils align head to v x 4 tall formIng two staggered rows Coned o 4 d 39 Fouru protofibrils come together to form a 00 md a E 9 39 80A microfibril 39 Microfibrils come together to form 2000A ommmal g a macrofibrils tails 39 5Q gt E a t E 0 keratin is rich in cysteine residues forming a 3 both inter and intrastrand disulfide bonds 1 a 39 Disulfide bonds contribute to mechanical My a robustness and rigidity h 39 Over 30 types of keratin have been identified Not uniformly distributed different keratin have different preferred locations 0 The structural protein synthesized by spiders for webs and by silkworms for cocoons 0 The broin proteins such as silk are thought to consist of extended arrays of antiparallel Bsheets with irregular regions of unknown structure linking the strands 0 The Bsheet regions consist of a repeated sequence GlyAla2GlySer GlyAlaAlaGly Ser GlyAlaGlyAlaGlygTyr 0 May be repeated 50 times resulting in silk polypeptides with massed ranging from 300 and 400 kD 0 GlyAlaSer residues may make up 85 of total amino acid compostion Gly 45 Ala 30 and Ser l5 0 The order of Gly and AlaSer residues 39 suggests that the Bsheets have a predominantly Gly residues on one face of the sheet and AlaSer on the other Silk Fibroin 035 nmt39 The sheets are thus stacked on top of each other with the Glyrich surfaces 057 nm packed against each other with the AlaSer faces packed similarly Larger side chains can t be accommodated by the tight backing between the sheets and are usually located in regions linking the Bstrands These regions have not been clearly de ned The silk polypeptide is stored initially as a concentrated concentrated aqueous solution with a structure resembling random coil Silk is extremely strong because the polypeptide strands in the antiparralel 3 sheets are in a fully extended conformation Colla en Three i intertwined Collagen an extracellular protein found In all multlce lular helices organisms Found in connective tissue such as tendons cartilage the organic matrix of bone and the cornea of the eye Collagen is the main constituent of higher animal frameworks bones tendons skin ligaments blood vessels and supporting membranous tissues Collagen peptides are distinct in their repetitive 5 sequences every third res is Gly ie GlyXaaYaan Pro residues are frequently in the Xaa and Yaa positions Endon views of collagen it Many of the Pro and Lys residues in the Yaa position are hydroxylated it Nearly l3 of the amino acid residues in collagen are Gly while l530 are Pro or 4hydroxyprolyl Hyp residues The reason for the repeating sequence becomes apparent from the structure of collagen NH HO OH 5 X 3 7 I Z x A 7 c r r 1 1 1 1 residue residue residue Three intertwined Collagen The reason for the repeating sequence becomes apparent from the structure of collagen Three collagen polypeptide chains lefthanded helical conformation wind around each other aligned in parallel in a righthanded fashion to form a triple helical coiled coil Every third residue must pass through the center of the triple helix which explains why every third amino acid residue must be Gly Endon views of collagen The polypeptide chains are staggered so that residues are not all in a single line Gly Hydrogen bonding occurs between the NH of each Gly and the carbonyl oxygen of a residue in the Xaa position Pro and Hyp confer rigidity on the chain and the wellpacked helical structure gives collagen great tensile strength Collagen assembly is aided by the presence of ICC to 300 residue prosegments that are cleaved following assembly Three intertwined helices Collagen Tropocollagen most fundamental structural unit of collagen isolated from young animals Tropocollagen is a triple helix assembled from three polypeptide chains of similar lengths l000 amino acid residues Therefore tropocollagen is a rodlike protein l4 nm x OOnm with a molecular weight of 285 ltD Endon views of At least 30 genetically distinct polypeptides collagen comprising l9 structural types of of collagen occurring in different tissues Four major classes have been identi ed Type Function amp Location Type i Chief component of tendons ligaments and bones Type ii gt50 of cartilage protein also in notochord of vertebrate emb 05 Type iii Strengthens walls of hollow structures arteries intestines and uterus Type iv Forms basal lamina basement membrane of epitneiia O TH LW TH H pl TH I oxidase I I Cross Linkin if 339 wi g 1 Aiiwne aldol ondensation 0 Crosslinking begins with the fgtCH1 oxidation of Lys or Hyl by lysyl HN ol 3 quotll oxidase a copperdependent EH39CHICHI H39cHI39CHICHI fH CO CH OC enzyme that OXIdIzes the Eamlno 4 g 4 group of Lys or Hyl to an aldehyde H forming aysine iii TH39N E CH1 0 Initial crosslinking step involves N aldol condensation between two m NJ ll oxidized LysHyl residues lHCHxCHzfHlHCHlcHI CHz lH co H oc H 0 Further crosslinking can occur with quotlquot 0 1 THNi His and LysHyl residues tri and OCHI tetrafunctional crosslinking k kw base ll le 7t 0 Crosslinking can occur both intra Wm aweH1CHlHCH1CHlCH1 H and intermolecularly OH x lo H o I I 439 N 39l39quot HxNCHlCHCHlCHl CH 5H 0 Collagen crosslInkIng IS progresswe O H04 and is tissue dependent Generally 39l39quot igzo crosslinking increases with age n HlE 0 Some types of collagen assemble to form distinctive banded brils 0 Driving force for bril formation is related to the hydrophobic interactions associated with the resulting ber 0 The packing of the collagen molecules results in the banding pattern observed in collagen brils gaps between aligned collagen molecules 0 Crosslinking collagen is inter and intramolecularly crosslinked near the N and Ctermini formed between Lys and His residues from four chains 0 Collagen brils are organized in tissues based on the function and the nature of the stress experienced by the tissue 0 Oglycosylation mostly glucose calactose and disaccharides of Hyl residues primarily in the gap regions Muscle ContractionAictin and Myosin gig 0 Muscle contraction takes place as a a result of the sliding of two interdigitating brous proteins Thick laments myosin Thin laments Actin Organized into 23 pm contractile units called sarcomeres Titin is another brous protein component of sarcomeres Titin is thought to help measure the length of the sarcomere Muscle contraction driven by ATP hydrolysis mm mm mun Hm Numml mun Figure H1llriiik u mi unilu W 1hr mumquot a I llun umumui tin uni mi lulu Hl u i 1 in mm nn ninncnh ir Ntw rii mi iii noi l Introduction to Protein Structure 2nd Ed p29 Muscle Contraction I The sliding of the thick and thin laments in the sarcomere is brought about by the motions of crossbridgesquot Crossbridges extend from the myosin laments and interact cyclically with the thinner actin laments driving the sliding of the two laments by a kind of rowing action The process is driven by ATP hydrolysis couples hydrolysis with confor mational changes in the the cross bridges and bindingrelease of actin Myosin in the thick laments is a brous protein with chains coming together forming coiledcoil structures Actin in the thin lament is a brous protein formed by linking together globular subunits Actin and Myosin llmi li39illw39rll um himn 1 no ligurv N we lirllnr nmmm mmlrl n mu m nimn Thi mm nun uni un39osn nith lH unan mmmmg l ytui row in n Iuiuulm mow u m yum Inuluml minim I m Introduction to Protein Structure 2nd Ed p29292 Muscle Contraction 0 A swinging crossbridge model has been proposed to explain movements 0 the heavy and light laments contraction Originally it was believed that myosin crossbridge binds actin with the crossbridge oriented at a 90 angle relative to the lament axis The crossbridge then shifts to a 45 angle relative to the lament axis Followed by release of actin lament 80l00 movement per cycle of ATP hydrolysis consistent with the swinging crossbridge modelquot Actin and Mvosin llim nimm um lilumrul mil quotwill i quoti mi and aquot in slhlillk39wliim rnnnamnn mum u u mm mm Introduction to Protein Structure 2nd Ed p29292 Muscle Contraction 0 HE Huxley et al observed lowangle xray ber diffraction patterns in living frog muscle revealed a series of layerlines arising from the helical arrangement of crossbridges distance between crossbridges was 435 A Using a synchrotron radiation source Huxley et al were able to observe the expected changes in diffraction data over time that were consistent with the swinging cross bridge model Actin and Mvosin iiiul lilmwiir mm 39iliimrul e liming ninmcm nin m m mu in mi Introduction to Protein Structure 2nd Ed p29292 Structural Studies and Actm and Myosm 0 Structural studies of both myosin and x x 39 actin have shed light on many long standing questions concerning cross bridge motion 0 Fibrous actin Factin is a helical polymer assembled from globular 375 residues polypeptides Gactin 0 Gactin is comprised of4 domains 2 domains resemble domains associated with ATPase active sites Gac ith boundA rp ppm ZHMP 0 Factin helix has l3 Gactin units m if assembled to 6 turns of the helix repeating every 360A 0 Ken Holmes et al used this information to determine the orientation of the Gactin subunits in Factin bers using 39539 39 V V iii in ray ber dl racmon Introduction to Protein Structurean Ed p 292 Structural Studies and Actin and Myosin Nii Hilillm yum mil 0 w u mum hold dmiris 1 ill C rlvrmivms H0 um 0 Myosin is a dimeric protein consisting of two heavy chains and four light chains I400 long Cterminal tail consists of parallel 0helical coiledcoil 2 helices Two Nterminal heads 0 Myosin can be fragmented to provide a fragment that contains the two light chains Nterminal head and portion of tail from one heavy chain from chicken myosin Introduction to Protein Structurean Ed p 294 Structural Studies and Actin and Myosin HJllQ39l milul hm u elm s light hams 2 nm C V Clermiuus l 7 50 nm 0 The structure of the head fragment from chicken myosin has been solved The head contains a 7stranded 3 sheet Oi3 motif and numerous associated helices Forms cleft containing both the actin and nucleotide sites These sites lie on opposite sides of the sheet ATP binding site changes conformation depending on whether ATP or ADP is bound Introduction to Protein Structure 2nd Ed p 294 Structural Studies and Actin and Myosin The cleft separates two parts of the head 50K upper and 50K lower domains The tail has a long extended X helix formed by the heavy chain and binds two light chains 0 A lowresolution model has been constructed based on structures of Factin and the myosin fragment Cleft in myosin extends from the ATP binding site to the actin binding site In the model proposed by Milligan the Cterminal helix the lever arm is joined to the bulk of the molecule via a converter domainquot W Introduction to Protein Structure 2nd Ed p 295 0Domains Continued ntroductjon to Protein Structure Chapter 3 Large amp Comnlex nzymu have baaquot mu m conum domzmx x zrg x 300400 renduax canmtmg a more mquot 20 0 ex pic a doughnut or Mum Obxervad My mum mmmmm Wm Nzermm 450 renduax mm man 2 mucmr conummg 27 a ux 7mg mm H m mm phy re My mum xpec cwy 0heica Domains c N 5mm 5 caHWctvansg WDS ase 70m wax 15m The Globin Fold I The Globin fold is one of most important x structures I Found in large group of proteins myoglobin hemoglobin phycocyanins and algal lighharvesting centers I Relative orientation of interacting helices in the globin fold is distinct from that found in coiledcoils and helical bundles I Globin fold is a bundle of 8 helices labeled AH linked by relatively short connecting loops I Orientation and positioning of helices creates a pocket for the active site Sperm Whale MyoglobinPDB IVXF I In myoglobin heligtlt length ranges ro residues in helix C to 28 residues in helix I Majority of packing interactions occur between pairs of helices that are not sequentially adjacent with helices G and H being and m exception I Geometry of helix orientation in globin fold reflects packing scheme ridge and groove helices pack at 9 50 relative to each other Sperm Whale Myoglobin g v f 51 o 9 wquot i quotA kalx a4 19 Pb 0 Globin Fold has been Preserved emoglobin and myoglobin hgure prominently in our th39e s H understanding ol protein o Globin domains have been observed in proteins lrom diverse organisms mammals insects and plants Demonstrate varied amino acid sequence homology m 99 Provides and example ol conservation ollold o Hydrophobic interior is preserved in each case 0 in comparing sequences ol proteins with globin lold Se quence conservatlo n and slzcom pensato ry mutation in hydrophobic core are not imponznt Strong prelerence lor hydrophobic residues in buried positions 59 such positions in yobin such conservation in surfaceexposed positi nx Helix movement can accommodate mutation th buried pos39 ons Loop flexibility allows one or more helices to xh t without musing other helices to shilt as well Important to maintain integrity ol active site Hemoglobin Hemoglobin is a tetrameric protein consisting ol two dillerent kinds ol polypeptide chains 2 X subunits and zrs subunits Single erythrocyte contains hemoglobin at a concentration o 3 mgml Sicklecell anemia is an inherited disease involving a GluAVal mutation at a surlace position 6th residue in hemoglobin 5 subunits The position is solvent exposed tetrameric protein Mutation promotes polymerization ol deoxygenated hemoglobin within erythrocytes does not promote aggregation ol oxygenated lorm Hemoglobin aggregate nbers give erythrocyes their characteristic cycle shape Lethal lor homozygotes lor disease Gives increased resistance to malaria 0U B Structures Introduction to Protein Structure Chapter 4 CUB Structures 0 In the known proteins CUB domains consisting of a central parallel or mixed B sheet surrounded by C helices are the most common domain structures In most CUB domains bindingcatalytic sites are formed by loop regions 0 Three main classes of CUB domains CUB barrel central B sheet forms a barrel structure with all B strands twisting around like barrel staves Connecting 0 helices are on outside of the barrel N Consists of a twisted B sheet flanked on both faces by connecting 0 helices 5 Leucinerich motif contain repetitive regions with a conserved pattern of Leu residues Consists of a central twisted B sheet with 0 helices on outer surface horseshoe fold All are assembled from BCB motifs Introduction to Protein Structure 2nd ed pp 48 56 0 B Structures 0 All 0B structures are assembled from B0B motifs 0 Two fundamentally different ways that two B0B motifs can be arranged to form a 4stranded parallel B sheet I In the barrel and horseshoe structures consecutive BtXB motifs connected in such a way that the motifs are similarly oriented N In the opentwisted sheet consecutive BtXB motifs are connected in such a way that the second BtXB motif is flipped I and turned around resulting in X helices being placed on both f faces of the twisted B sheet 0 Nearly all B0B motifs are righthanded mm i i Introduction to Protein Structure 2nd ed pp 48 49 Introduction to Protein Structure2nd ed pp 48 O B Barrel ln 0B structures where the strand order is l 2 3 4 all connecting helices lie on the same face of the sheet Such an arrangement would leave the other face of an isolated open twisted sheet exposed to solvent Usually forms a closed barrel assembled from twisted B strands with connecting 0 helices on the outside of the barrel More than four B strand are required to form a closed barrel usually 8 strands and sometimes l0 strands In almost all 0B barrels the crossconnections between strands consist of 0 helices with there often being an additional 0 helix after the last strand 39 At a minimum of 200 residues the eightstranded 0B barrel is one largest domain structures I Very common structure found in many different proteins Amino acid sequence varies significantly but the core DUB barrel structure is conserved N Depending on protein connecting loop regions can have varied lengths and amino acid composition in some loops may fold into distinct domains Triosephosphate lsomerase from Giurdiu lumbiu PDB2DP3 CUB Barrel Core 0 In we barre s hydrophobwc swde chem frorn tne u hehces pack agamst hydrophobwc swde chams on tne outer surface of tne e sneet a As 5 charactemmc of an 8 sheet tne swde chem of consecutwe feswdues are omented on oppos te faces of me e sneet a Every second reswdue commbutes to tne hydmphobm surface tnat packs agamst tne hehc s o Otner swde chams of tne e strands are dwrect d mwards towards tne hydmphobm core of tne oarreL o Packng meramons between a hehces and e strands are dofmnated by reswdues vntn orancned hydmphobm swde chem th I and Laccount for 0 of reswdues MIN 8 strands nthe pan1H3 Bsheets o I the baNeL Formatwgm yspacked hydmphobm core Pam and charged feswdues K K and Q termmam some of tne strands The Wdrophobwcahpmmc pomon of tnese swde chem of feswdues m tnese posmons conmbute to hydrophobwc core ruvate Kinase a Ax known Ssstranded we barre domams nave enzymanc funmonsfsuch as39 own pf pa yucchzndex and o goucchzndex o In some cases the we barre domam s tne domwmnt s V structum e ememf m otners tne po ypep de cham forrns t mu twp e domams m addmon to tne ms baNeL Q o Even m tne mu twdomam protems enzymzmc amwty s usuauy assocwated wwth tne we barre domn 5ucn s tne case w n pyrmte kmase Pyruwte kmasef wmch cata yzes tne transfer of a pnospnoryf group from phosphoeno pymvate to ADE s fo ded mm four dwsnnct domams o In pymvate kmasethe we barre domam s mvo ved wwth substrate bmdvg and and prowdes catach groups a A domam conswstmg of an armpamHe e sneet he wthm one of tne maps of tne centra we barre Double Barrels and Gene Fusion PRA isomeraselGP synthase from all consists of a single polypeptide chain that folds to form two otB barrel domains PRAisameraselGlgtsynthase 4mm E cali PDB pii o The bifuncu39onal enzymes catalyze steps inthe synthesis of tryptophan The cterminal PRAI domain catalyzes the conversion of 5 phosphoribosyl anthranilate PRA to lo carboxyphenylamino ldeoxyribuose 5phosphate CdRP s two consecutive The Nterminal domain catalyzes the ring closure in CdRP to form indoleBglycerol phosphate lGPi ln Bacilus subtilis these reactions are catalyzed by two separate enzymes which have amino acid sequences homologous to the corresponding regions of the bifunctional enzyme 0 The analogous protein in Neuruspura crassa catalyzes three reactions inTrp biosynthesis The enzyme co t in two catalytic odB barrel domains similar to those in the other organisms but also contains a third catalyu39c domain 0 Reflect differences in in genome organization in these organisms Evolution of New Enzyme Activities in lmllnllilt n NW viuuil uni it g mm 048 barrels provide insights into the process arising in the evolution of new enzyme activities m Hi ll lit I n 0 Evidence suggem that new enzymes m H V a 39 7 arise from divergent evolution from W mmmWHWW preexisting enzymes 0 39 o Greg Peuko et al studied oiB barrel C x m enzymes involved in the conversion of f mandelate to benzoate a relatively 039 a quot I recently evolved synthetic pathway 39T l The frst enzyme in this pathway mandelate racemase catalyzes the interconversion of the two optical isomers of mandelate via abstraction of the proton at the chiral center producing an enoIic intermediate Petsko found that mandelate racemase is with its otB barrel is very similar to an m on e lactonizing enzyme which catalyzes a very different chemical reaction but the mechanism of muconate lactonizing enzyme also involves proton extraction from a carbon atom The amino acid sequences of these enzymes showed 26 sequence identity indicating an evolutionary relationship Evolution of New Enzyme Activities on quotlllmft39l it liliflllllai Main Main iilmnmimlr wmim iii 2 H u k39H Ho The amino acId sequences of these quot 39 enzymes showed 26 sequence identity indicating an evolutionary relationship H lCH u ML C 0 1 39 039 Cf OH H Comparison of the sequences of the W quotwrmmrvlur39vnirirmMilquotw two enzymes revealed signi cant similarities in their active site residues 4quot involved with proton abstraction while the positions involved in substrate recognition differed 39 i D 39H quot39 l l 39 l Suggests that mandelate racemase likely evolved from a preexisting enzyme that catalyzes proton abstraction and formation of an intermediate Mutations over time have altered substrate speci city such that the enzyme now acts upon mandelate Preservation of the fundamental chemical process is important for preservation in the evolution of the new enzyme speci city however can be modi ed in the process Results suggest that speci c enzymes evolve from relatively nonspeci c enzymes which may have existed earlier in evolution or have arisen through random genetic rearrangements in more speci c enzymes Gene duplication must occur at some point Leucinerich Motifs Leucinerich motifs consist of tandem repeats of PDB 2bnh homologous 2030 residue righthanded Bloopa structures r Such leucinerich motifs have been identi ed in over 60 different proteins ranging from receptors cell adhesion molecules bacterial virulence factors and proteins involved with RNA splicing and DNA repair n ribonuclease inhibitor a polypeptide chain consisting of 456 amino acid residues has been arranged to form a tandem repeat of IS leucine rich motifs 2 flavors type A with 29 residues and type B with 28 also contains two short regions with nonhomologous sequences at the termini Both type A and type B demonstrate similar pattern of Leu Sequential Bloop a repeats are joined together in similar way to those in the CUB barrel structures The strands form a parallel 3 sheet interior surface of a curved open structure aquothorseshoe A Introduction to Protein Structure 2nd ed pp 56 Leucinerich Motifs With the B strands lining the inner surface of the 0 PDB bnh helices adorn the outer surface of the open curved structure 0 helices are aligned antiparallel to the B strands Hydrophobic packing between outer face of B strands and the inner surface of the 0 helices Unlike OIB barrels the inner surface is exposed to solvent The Leu residues of the leucinerich motifs form the hydrophobic core between the strands and the helices Leu residues at positions 2 5 7 l2 20 and 24 are invariant between type A and Type B repeats Examination of more than 500 tandem repeats from 68 different proteins indicates that residues at positions 20 and 24 may be other hydrophobic residues ie He and Val Ribonuclease inhibitor has been used as the basis for constructing plausible models for other proteins with leucinerich motifs K Introduction to Protein Structure 2nd ed pp 56 OIB Twisted Open Sheet was r f n OIB twisted opensheet structures the connecting 0 helices are positioned on both faces of the B sheet A This arrangement Precludes formation of a barrel structure N Results in two adjacent B strands in the interior of the sheet Le BI and B4 whose connections to the flanking B strands are on opposite faces of the sheet W Oi helices are packed against both faces of the sheet with each B strand contributing to hydrophobic side chains to pack against Oi helices in two similar core regions one on each side of the B sheer an e of the connecting loops from one of these two strands goes above the sh39e39et while thevthe other lo op goes below the sheet Pattern of Connecting segments results in crevi ce outside the edge of the B sheet between these loops BI and B4 Almost all binding 9 in DUB tWistjed sheetjs occur in such crevicesfou nd at39the carbpxy vi edge of39the she et Introduction to Protein Structure 2nd ed pp 48 57 CUB Twisted Open Sheet Topologies 0 While odBbarrel domain structures show the same basic armngement of 8 olt helices and 8 3 strands open Bsheet structures show a variety of topologies 0 Because the 3 strands form an open twisted 3 sheet there are no geometric restrictions of the number of stmnds generally ranges from 4l0 strands 0 Two consecutive strands joined by a crossover connection need not be adjacent to each other in the folded 3 sheet BmB motif with strands being adjacent is preferred 0 Allows for mixed paralleantiparallel 3 sheets that incorpomte 3 hairpin connec ions Active Site Prediction in CUB Structures 0 In almost every known MB twisted open sheet containing protein the active site is located at the carboxy edge of the sheet 0 Functional residues reside in the loops linking the Cterminal ends of the 3 strands to the Nterminal ends of the olt helices 0 Analogous to the localization of active site residues in DUB barrels 0 Unlike DUB barrels open DUB structures cannot form unnelshaped active sites Instead they form crevices at the e ge of the sheet 0 Crevices are formed when there are two adjacent strands with connections that are on opposite sides of the 3 sheet Immdu on 2 Protein Structure 1nd ed pp 57 Active Site Prediction in CUB Structures Crevices are formed when there are two adjacent strands with connections that are on opposite sides of the 3 sheet Position of crevices is determined by 3 sheet topology and can be predicted based on a topology diagramt Crevices occur when the strand order in the sheet is reversed Crevices occur where connections from the carboxy ends of two adjacent strands in a topology diagram go in opposite directions referred to as topological switch points The ability to fairly reliably predict the position of active sites in all structures is in contrast to the other two main classes structural classes xhelical proteins and antiparallel 3 proteins No obvious predictive rules for these classes of proteins have been determined flfi l t Introduction to Protein Structure 2nd ed pp 57 58 TyrosyItRNA Synthetase AminoacyltRNA synthetases connect amino acids with its speci c tRNA in a two step process Initially the amino acid is activated by generating an amino acid adenylatet uses ATP Then the complex is attacked by the tRNA OH to give the aminoacyltRNAt The structure of the rst 320 residues of tyrosyltRNA synthetase reveals two domains one is an aB domain and the other is an 0 helical domain The aB domain is associated with binding ATP and the substrateTyr but the function of the 0 helical domain is unknown TyrosyItRNA Synthetase 0 On examining the topology of tyrosyltRNA synthetase The domain contains a 6stranded mixed B sheet Strand order of the parallel component is 652 3 and 4 A switch point occurs between strands 5 and 2 O Expect crevice at this point and likely active site 0 The active site has been determined by diffusing Tyr and ATP into the crystal 0 The tyrosyl adenylate binds in a cleft lined with loop regions 3847 at the end of strand 2 and l90l93 at the end of strand 5 O The substrate straddles the edge of the B sheet with the Tyr and adenine ends on opposite sides of the sheet Substrate makes additional interactions with other residues in the region surrounding the Intruduaz39urr ta Pruteirr Structure 2nd ed pp 60 creVIce Carboxypeptidase Carboxypeptidases are zinccontaining enzymes that catalyze the hydrolysis of polypeptides from the Cterminal peptide bond 0 The bovine enzyme is a monomeric protein 307 residues in length The protein consists of an open twisted OKIB structure with mixed B sheet Intruductr39urr ta Pruteirr Structure erd 0 Some of the loop regions are very long and curl around the central structure i ed pp 6 0 The bound zinc ion is essential to catalysis lt binds the carbonyl h oxygen to the substrate amide bond drawing electron density U 39 from the carbonyl carbon atom and facilitating cleavage of the IN u A a r r 2 v peptide bond 0 The four central strands of the B sheet strands 8 5 3 and 4 are parallel with a switch point between strands 3 an O The catalytic zinc atom is located in the crevice formed by the switch point at strands 3 and 5 It is coordinated by the side chains of Hi559 Glun in the loop at the end of strand 3 and His95 at the end of strand 5 0 ln carboxypeptidase the crevice accommodates the activesite zinc ion and loop regions directly adjacent to the B strands are not directly involved with substrate binding B Structures Introduction to Protein Structure Introduction to 3 Structures Armpde s munqu compm m xacend zrg group a V WWW WWWMW m m n m M 0m mm wan comm ME mm rnngad nuparzHL umeyhrmmg we mm 33 mm m pickzgzmxt Bach other pa mam mum0pm w s bzna x m s mucturex m 33 mm H mm and Wm m mm 7 packad gzmxt nah other u uxmw 72mm UV 2 mum mum Armpde s mucturex gananHy have 2 or a Nydrophob c m chm mm m mama m xurfzc mm by mm mm m bow and m mm m numbar a mu topohgxex or mm 2 nup nH 33 mm mm mcruaxexex thn m number 6 mm mm n u xurpnxmg m m numbar a topa egmx that have ban 3 w x duverymnH wnhmon xtructurexmhngwnhmzhw groupx a cammonlnm zr topo ogmx Common B Structure Topologies 0 Most B structures fall within a few groups of commonsimilar topologies l upanddown barrels 2 Greek key motif 3 jelly roll barrels UpandDown Barrels 0 The simplest B structure topology is the up anddown barrel assembled from successive B hairpins This arrangement allows considerable structural and functional versati ity V 0 Retinalbinding protein REP is a good example of a upanddown B barrel containing protein 0 RBP consists of a single 82 residue polypeptide chain It is responsible for the transport of retinol vitamin A from its storage site in the iver to vitaminA dependenttissues ll stoichiometry 0 RBP contains a Bbarrel core consisting of 8 B strands aligned antiparallel to each other and a short Cterminal helix that packs against the outside of the barrel namppss UpandDown Barrels RBP contains a Bbarrel core consisting of 8 B strands aligned antiparallel to each other and a short Cterminal helix that packs against the outside of the barrel The B strands of the barrel are curved and twisted and one end of the barrel is open to solvent the other end of the barrel is closed by tight packing of hydrophobic sidechain packing Hydrophobic retinol binds at the open end of the barrle its hydrophobic tail inserted into the hydrophobic interior of the barrel The threedimensional structure of the apo protein does not differ signi cantly from that of the protein A K with bound retinol ll l A large part of the RBP surface consists of side A chains from residues in the B strands As a result the strands show an amphipathic pattern of amino acid side chains hydrophobic residues directed into the barrel core alternating with polar and charged residues displayed on the outer surface of the barrel The structure of the protein also indicates that the barrel is build up from two B sheets Introduction to Protein szmame 2nd ed pp 68 Introduction la ngein Structure 2nd ed pp 7 2k Neuraminidase Neuraminidase from influenza virus example of an upanddown B sheet arrangement The enzyme removes sialic acid residues from carbohydrate chains on viral hemagglutinin and glycosylated cell membrane proteins facilitating the release of progeny virions from infected ce s is another The arrangement and packing of the B sheets in neuraminidase is dramatically different from that observed in RBP The B sheets in neuraminidase do not form a simple barrel instead forming 6 small 4 stranded sheets A H arranged in a pattern resembling the blades of a g propeller Ii 7 H Jl llj u in 2 i 539 n The loop regions between the B strands in the middle of the propeller structure form the active site Neuraminidase is a tetrameric proteins consisting of 4 identical subunits each chain 470 amino acids in length The soluble head compoonent can be proteolytically separated from the stalk membrane anchor Neuraminidase Each of the four subunits of the tetrameric neuraminidase head is folded into single a domain assembled from six closelypacked similarly folded U1 1 quot m H fourstranded antiparallel B sheet 39 1 l 1 6 a a m e a The strands in the B sheets have a large twist such that the direction of the lst and 4th strands in each sheet are oriented at 90 angles relative to each other The tetrameric protein consists of 600 residues and is composed of four identical polypeptide chains each of which is folded into the propellerlike super barrel structure to the right Introduction to Protein Structure 2nd ed pp 772 Neuraminidase The 6 B sheets within each subunit are connected to each other by a loop that runs from the end of the 4th strand in the preceding sheet across the top of the subunit to theIl rst strand in the next sheet In the last 6th B sheet in the subunit the outermost strand the 4th in the other sheets is the overall rst strand in the chain and is not connected to strand 3 as is the case in the other 5 sheets The loops connecting the 2nd and 3rd strands in each sheet also lie at the top of the structure The B sheets are arranged cyclically around the axis of the propeller and these loops at the top of the barrel Iu Adlw SIIL Collectively these loops form a wade funnel shaped pocket containing the active site Introduction to Protein Structure 2nd ed pp 772 Y crystallin and Do Crystallins are located in the lens oi the eye and they contribute to the optical properties oi the lens Three classes oi crystallins have been discovered ix an v and t ere may be more The ix and p crystallins are heterogeneous assemblies oi diiierent subunits oi diiierent subnits while ycrystallins are monomeric l7oresidue proteins 0 The crystal structure oi a ycrystallin reported b h 7 Bl ndell et al reveals that t e protein is lolded into two distinct domains that are similar in size and are connected by a flexible tether The secondary structure oi the Nterminal domain is dominated by p strands which are arranged into two antiparallel sheets Sheet 2 l4 and 7 amp sheet 5 5 s and 3 While strands 5 and 7 are adjacent in sequence they share no hydrogen bonds lmrodutz 4 Y crystallin and Doma39 The highlighted Nterminal domain consists oi two iourstranded 5 sheets The arrangement oi the p strands oi this domain is illustrated in the provided topological diagram The two 5 sheets in this domain are packed against each other iorming a distorted barrel structure Strands 2 3 and 4 iorm a Greek key motii as do strands 56 7 and s The Nterminal and the cterminal domains have identical topologies and very similar structures Ycrystallin and Domains The Nterminal and the Cterminal domains have identicalQ topologies and very similar structures While the structural similarity of the two domains is not immediater obvious from the crystal structurethe similarity in their topologies is clearly evident in the topology diagram In the folded protein the polypeptide chain is divided into four consecutive Greek key motifs grouped pairwise in the tw domains Overlaying the Cu atoms of residues in the two domains sults in a mean deviation of ltZA indicating that the two domains are structurally equivalent u The motif structures within each domain superpose equally well but the have lower 1 h 39 m Similarity between the four Greekkey motifs suggests that they are evolutionarily related mm 2 1931 mm x um i Walter Gilbert in I978 suggested that genes for larger proteins may have evolved by the accidental juxtaposition of exons coding for speci c functions I v f i H mm 2 mu introduction to Protein Structure 2nd ed pp 75 Jelly Roll Barrels In antiparallel barrel structures built the Greek key motif one 0 connections in the quotquot motif crosses one end of the barre The jelly roll motif is a common motif in which four of the interstrand connecting segments cross the ends of the barrel The jelly roll motif is found in a variety of proteins including coat proteins of most 5 herical viruses concanavalin A a plant lectin and influenza virus hemagglutinin In the jelly roll motif the polypeptide chain has 8 B strands connected by loop regions 339 ru 5 umijillu mmr m law l u 1 M m Essentially the 8 B strands are arranged in a long lm h ll ll l39lmlllhllljltvll ll m antiparallel hairpin such that strand l is Hbonded if quot 39 with strand 8 and strand 2 is paired with strand quot 7 and so on irmhni my mm m min in p v uni m miw The extended hairpin is then folded into a barrel con guration with the B strands forming the sides of the barrel and the connecting segments crossing both the top and the bottom of the barrel introduction to Protein Structure 2nd ed pp 77 In the barrel structure antiparallel Hbonded B R B I strand pairs 8 27 36 and 45 are arranged such y o s that B strand l is adjacent to strand 2 strand 7 is adjacent to 4 5 to 6 and 3 to 82 W m m In the barrel structure all adjacent strands are a aligned antiparalleli This barrel arrangement results in two connecting u loops 34 and 78 crossing the top of the barrel and two 23 and 67 across the bottom of the barrel 1 The described arrangement is for a 8stranded jelly roll barrel but a jelly roll barrel can contain any 3 even number of B strands greater than 2 8 stranded barrels are most common Analysis of the Hbonding patterns in jelly roll barrels reveals that they usually can be broken down o shees with few if any Hbonds between strands belonging to the different sheets2 Barrel is distorted from the ideal with gaps separating two pairs of adjacent B strands in the barrel The B strands in jelly roll barrels are often arranged x I j r r 1 u i in two sheets that are packed against each other imradumar in Protein Strudure2nd ed pp 77721 Hemagglutinin ln influenza virus hemagglutinin mediates binding of viral particles to host cells recognizing sialic acid residues in cellsurface glycoproteins2 Wral particles bud from patches of the cellular membrane that contain hemagglutinin and neuraminidase Hemagglutinin consists of two polypeptides chains HAI and HA2 with one of the polypeptide chains HAI containing a domain with a jelly roll barrel motif Synthesized as a membranebound single polypeptide chain that is proteolytically cessed to afford 328 HAI and 22l residue HA2 peptides2 HA and HA2 are connected by disulfide bondswith HA2 anchoring the peptide to the cell membrane During processing hemagglutinin forms membranebound SYmmEEFiC trimerSA lntmductmn m Prmerr Structure 2nd ed pp 79 Hemagglutinin Each monomeric subunit is divided into a long and brous stemlike region extending out from the surface of the membrane wide a globular region at its tip The globular region is entirely contained widqin HA while both HA and HA2 contribute to the stem regionl Nterminal portion of HAI is located at due base ofthe stem The globular tip consists of a distorted 8 stranded jelly roll barrel comprising residues II626I of HAL The remaining Cterminal residues of HAI return to the stem region The major structural feature of HA2 is a hairpin loop of two x helices packed against each t odaen At the base of the stem is a 5stranded antiparallel 3 sheet a central strand from HAI flanked by pairs of strands from HAL lmmducnon to Protein Structure 2nd ed pp 79 Hemagglutinin The hemagglutinin timer is an unusually elongated protein assembly I35A long widq a crosssection between IS and 40 In the trimer the interface between the structure The trimer structure is further stabilized by interactions between due head regions of the subunits tiate infection viral hemagglutinin binds to binding sites are formed by the jelly roll domainsl lmmducnon to Protein Structure 2nd ed pp 79 Hemagglutinin 0 Sialic acid binds in the center of a broad pocket on the surface of the barrel in conjunction with a hydrophobic channel to accommodate large hydrophobic substituents at the C2 position on sialic acid Introduction to Protein Structure 2nd ed pp 79 8 l Hemagglutinin 0 Hemagglutinin has a second function in the infection of host cells I owe red membrane and endocytic vessicle membrane dramatic conformational change at lower pH s Viral particles bound to proteins at the cell surface are taken into the cell by endocytosis Protons are then pumped into the endocytic vessicles the pH of the compartment is As the pH in the compartment drops below pH 6 hemagglutinin still bound to the cell surface protein undergoes a conformational change thereby inducing membrane fusion viral Using monoclonal antibodies to speci c epitopes of the two chains HAl and HA2 it has been shown that hemagglutinin undergoes a Hull til i Introduction to Protein Structure 2nd ed pp 79 82 83 Hemagglutinin 0 Don Wiley revealed the conformational change that occurred by solving the structure formed by HA27 and HA23875 at acidic pH The loop region connecting helices A and C becomes helical forming one continuous helix ABC Region at CD interface loses helical structure becoming a loop connecting remainder of helices C and D Helix D shifts and packs against helix C The small X helices and B strands at the Cterminal end of the chain also shi The fusion peptide is attached to the N terminus of helix A At neutral and basic pH the fusion peptide is positioned about IOO A from the receptor binding site At low pH the repositioning of helixA places the fusion peptide in the same vicinity as the receptor binding site each subunit Note the low pH form appears to be more stable than the high pH form and does not revert to the high pH form when the pH is raised The energy required for fusion is likely stored up during formation of the hemagglutinin molecule H5 quotmin I m m my pH ymmwnl Introduction to Protein Stmrture 2nd ed pp 83 Parallel Bhelix Domains The B helix was rst observed in the bacterial enzyme pectate lyase by Frances Jurnak in I993 several FT proteins have since been found to contain Bhelix I structures In Bhelix the polypeptide chain is coiled into a wide helix formed by B strands separated by loop regions strands are aligned in parallel The twosheet B helix is the simplest form each turn consists of two strands and two loop regions n bacterial proteinases B helix consists of three turns of the helix essentially forming two threestranded parallel B sheets packing against each other with a hydrophobic interior The B sheets in a B helix are essentially planar lacking the twisting typical of 0B structures The basic unit of twosheet Bhelix structures contains 8 amino acid residues three in each strand and six in each loop Protein Stability and Folding Stability of the Folded Conformation Folded conformation is only marginally stable and can be disrupted by changes in environment heat pH increased pressure or addition of denaturants Protein denaturation need not involve changes in covalent structure and is usually reversible As the environment changes towards denaturing conditions initially the structure of small singledomain proteins changes very little May be increases in flexibility but overall structure is unchanged The protein then denatures entirely over a very narrow range This abrupt unfolding is indicative of a very cooperative transition The unfolding of most small singledomain proteins is reversible and equilibrium can be attained Many methods available for visualizingmonitoring unfolding Protein Folding aTwostate Phenomenon Protein loldingunlolding is a two state p enomenon with only lully lolded N and lully untolded U protein s tes being present Partally lolded states are very unstable relative to N and u states 0 For a twostate transition the equilibrium constant between N and u an be measure direttly lrom the avenge lmttion ol 1 n w H H unfolding cx in the transition region 39 quot irnlpiu39 or i in The value otlltq an be determinedwhen olt kl y r N is signmtantly dmerent lrom l or o in the N T a W tmnsitionre 39on 2 U N o Allows zkulztion ol AG under the set Km M J imam onditions dillerente in free ener U alt unludad U and N states 5 392 quot o When Kw LAG 0 provides a reterente AG GNV GU 7R 7ian tor taltulating A6 at other tempemturex AG AH r TAS vzn39t Holt analysis uses temperature dependente otllttq to estimate AHzmd AS AGO 01H T A5 in s in in in Protein Folding aTwogstate Phenomenon The heat upzdty CF is de ned as the luqu in entlulpy H with tempemture T o cP ot the unfolded protein cpu is greater than that ot the lolded protein CPN dilterente between the two is the Ac The cP ot proteins are dominated by the nonpolar surlate area expo d to water it is possible to taltulate A6 at any te pemture it one knows AcP and the AH m do so on w tein vennnsnoi at o AH is most easily estimated at the Tm Cr 6H 6T TGS 6T At high and low temperatures AG is M aT cw CAN MP dominated by the entropit tomponent AHT1 AHTl Aqua Ti Mm AHMTM Tm Aim Asa AiMl TTm ACPTmT T lnnm 1 Protein Folding aTwostate Phenomenon Chemical Denaturation 1 Chemical denaturants such as urea guanidinium N k U chloride or guanidinium thiocyanate can also be 2 used to determine thermodynamic parameters N 1 fora protein Keq U fraction of Chemical denaturants effectively increase the 0 Egggned solubility of hydrophobic sidechains decreasing hydrophobic contribution to stability of the folded protein AG GN 39 GU 39RTaneq As the denaturant concentration denat increases Keq shifts towards unfolded AG AH TAS AG under normal conditions RT in the absence of denaturant can be estimated by extrapolation AG0 usually between 5 to l0 AG AGO mdenat kcalmol O NHZ Parameter m reflects the dependence of AG on i the denaturant concentration HQN NH HQN NH Urea Gaunidinium cation m is dependent on the the denaturant in question The Unfolded State Many proteins under strongly denaturing conditions have been shown to have properties consistent with random coil conformations Under less denaturing conditions this is not necessarily the case If interactions between different parts of the polypeptide are preferred over interactions with solvent then the chain tends to be more compact and less disordered than expected for a random coil While the physical properties of unfolded states produced under different unfolding conditions may differ they are energetically indistinguishable Dif cult to characterize the unfolded state of a protein because many conformations are possible and may be populated The moltenglobule state under certain conditions proteins have been known to demonstrate properties consistent with a molten globule state I More compact that random coil marginally less compact than folded state I Secondary content is similar to that in the folded protein I Side chains are in homogeneous surroundings in contrast to folded protein where interior side chains exist in different and distinct environments Many peptide bonds rapidly exchange hydrogen atoms wida solvent more so than in the folded protein Enthalpy of molten globule state is similar to that of the unfolded state different from folded state lnterconversion of molten globule state with fully unfolded state is rapid and noncooperative with the folded state is slow and cooperative Thermodynamics of Unfolding 0 e temperature at which different T proteins unfold can vary enormously 0 Most proteins denature at elevated tempera res Thermal denaturation is of intrinsic thermodynamic importance 0 Thermodynamic studies have mostly focused on the twostate transitions of single domain proteins because of their relative simplicity 0 Stability decreases as temperature shifts in eid1e direch Almost all proteins denaturation is only observed under circumstances at which unfolding occurs at an accessible temperature to him so aimsitstiioittpi h t o it t ii iii imiciiiiiti o lo itiei rml i inii iis imi iiiii i e s 2a ienpiiaiiiirl Mum mvism oi tit me Alsvus ii lubumawusmwli zmima it meow it ROD mini and mitts Oval to cl as us 2 amnsenulm ttmiotiaw a 2m mus itpti cnei s icnua as 2 mm oi i Calorimetry allows direct determination of AHmi and determination ofthe CP ofa protein in solution cP of the folded protein initially changes only slightly as MP T is increased Sh increase associated ing WI CP of the unfolded protein is greater than that of the folded protein difference between the two is the Acp P th P the folded re and posttransition regions are those of and unfolded states in mter respectiver temperature dependence of their and provide the enthalpies and entropies The cP of proteins are dominated by the nonpolar surface area exposed to a greater CP than does the folded state water the unfolded state has Therefore AcP reflects the buried nonpolar surface area in the unfolding olded protein that is exposed with The large of AcP protein unfolding results in there being atemperature where stability of the folded state is at a maximum Thermodynamics of Unfolding Physical Interactions The unique properties of proteins is inextricably linked to the complex threedimensional folded conformations they assume The threedimensional folded conformation is the result of many simultaneous noncovalent interactions between different parts of the protein and with the environment These interactions are the result of a limited set of fundamental noncovalent forces The complexity of water and an aqueous environment limits our understanding of proteins Protein Stability Native proteins are only marginally stable under physiological conditions The free energy of denaturation is only 0l mad kcalmol for each amino amino acid residue The folded conformation of a l00 residue protein would be l0Kcalmol more stable than the unfolded state energy required to break a hydrogen bond is 2 l0Kcalmol M IAAG increasing Stability lgt G The threedimensional folded conformation arises through a delicate balance of stabilizing and destabilizing forces folded 3 The observed stability of the folded protein is the result of a very small difference between very large but compensating factors enthalpy and entropy both of which are temperature dependent The enthalpic and entropic contributions vary similarly and compensate each other This results in the free energy being relatively small difference between the two Forces Stabilizing Macromolecular Structure 0 Noncovalent interactions are key biological forces Electrostatic Forces gt lonic interactions gt Van derWaals gt Hydrogen bonding Hydrophobic interactions 0 These forces are transient in nature 0 Several factors influence the strength of these interactions 0 Individually all are weak CC bond 80 Kcalmol but they add up and collectively can be very strong ShortRange Repulsions Repulsion eventually occurs between two molecules or atoms as they approach each other Repulsion invariably arises as they molecules atoms become near enough for their respective electron orbitals begin to overlap Repulsion increases enormously because the electrons on the different molecules cannot occupy the same space at the same time increasing exponentially with the inverse of distance Because repulsion rises so steeply it is possible to consider moleculesatoms to have de nite dimensions with de ned volumes van der Waals radlus van der Waals radius is based on smallest distance that can exist between two nonbonded atoms in the crystalline state Accesslble surface area provides a more practical concept of surface for proteins and other biomacromolecules described by center of a water molecule with radius l4 in van derWaals contact with the molecule Electrostatic Forces Point Charges All intermolecular forces are thought to be essentially electrostatic in origin The most fundamental noncovalent interaction would therefore be the interaction between electrostatic charges Coulomb39s Law vacuum ZAZB 82 0 Coulomb39s law describes the interaction between two point AE r charges in a vacuum AB 0 It describes an interaction that is effective over relatively a the charge of an eIectron lon distances g ZA number of charges on A 0 For other IenVIronments such as In solution the 25 number of charges on B electrostatic Interaction Is modulated by other Interactions rAB distance between A and B 0 ln homogenous environments the electrostatic interaction is diminished by the dielectric constant 0 t e medium dielectric constant of water 80 0 At short distances molecules and atoms cannot be treated Adjusted for environment as point charges 2 0 Electrostatic effects in proteins involve changes in their AE ZAZB S ionization tendencies pKz values D rAB 0 Interactions between very close oppositely charged groups I I in proteins usually involve not only electrostatic D d39eleCtr39c consmnt interactions but also some degree of hydrogen bonding o Electrostatic Forces van der Waals 0 A molecule does not need to have a net charge to participate in electrostatic interactions 0 Electron densities can be localized if covalently linked atoms have different electronegativities Atoms with a greater electronegativity have a partial negative charge Atoms with a lower electronegativity have a partial positive charge 5 0 The separation of charge in a molecule determines its dipole moment up corresponding to the magnitude of the separated charge and the distance d by which it is separated ElectronegatIVIties 0 The dipole moment has directionality as well as magnitude D Zd Efcfggr atoms Dipole Moment P 3945 39 0 The peptide bond which has partial double bond character N 298 exempli es this polarization The oxygen has a partial C 255 negative charge and the NH group a partial positive 5 253 H 2 l3 Dipoles interact with point charges other dipoles and more complex interactions Electrostatic Forces van derWaals Dipolar interactions are weaker than those between ionic ml immumsmwmMamide groups ThIs Is due to the fact that both attraction and repulsion occur between the two separated charges The strength of dipolar interactions drops much more l abruptly with distance than is the case with the interaction with ions inversely with d3 my blamelamina mmlemyevmhuns Interactions involving dipoles also effects the dipole charge distribution wit in t e interacting molecules Polarizability describes the disposition of a molecule to have its electron charge distribution influenced by an applied electronic eld m Lennon min yam Polarizability reflects how tightly electrons are held by the nuclei Generally larger atoms are more polarizable gl 9 An induced dipole always interacts favorably with the inducing eld However this interaction is only half of that which would have occurred had the dipole already existed Electrostatic Forces van derWaals All atoms an molecules attract each other even in the absence of char ed groups as a result of mutual interactions and induced polarization w lnveracl m mmquot uwmm mm These are week interactions only effective at short distances varying with d39s Can arise from interaction between 0 Two permanent dipoles 0 A permanent dipole and an induced dipole 0 Two induced dipoles London dispersion forces London dispersion forces complex interaction Essentially an om or group may have no net dipole but may have a transient hquot midwsmmnwm dipole resulting from temporary asymmetry in the distribution of electrons This transient dipole can similarly polarize nearby 3 neutral atom synchronization of electron flow and distribution in neighboring atoms and groups Interaction becomes insigni cant at distances greater than 50 Van der Waals interactions are often represented by an energy potential as a function ofdistance d The optimal distance for the interaction oftwo atoms is usually 0305A greater than their combined van derWaals radii Electrostatic Forces Hydrogen Bonding A hydrogen bond occurs when two electronegative groups compete for the same hydrogen atom In such interactions the H atom is formally attached to the donor atom via a covalent bond and interacts favorably with the acceptor atom The main component of the hydrogen bond is an electrostatic interaction between the dipole of the covalent DH bond H has 5 and the 539 of the acceptor atom The electrostatic and covalent elements of the hydrogen bond make it energetically favorable for the three participating atoms DH and A to be collinear This is the most common arrangement however some deviation from linearity is observed The lengths and strengths of hydrogen bonds is dependent on the electronegativities of D and A The greater their electronegativities are the shorter and stronger the hydrogen bond The predominant hydrogen bonding in proteins occurs between the CO and the N H groups of the peptide backbone 639 6 639 DHA Common Common Hdonors Hacceptors N H O O H O S H N C H 5 S N H I Z p H39 I N Hydrophobic Interaction Electrostatic hydrogen bonding and van der Waals interactions between two molecules in an aqueous environment are not particularly favorable due to competing interactions with surrounding water molecules water are not as favorable In the case of hydrophobic surfaces interactions with surrounding The relative absence of favorable interactions with surrounding water molecules increases the favorability of interactions among nonpolar groups themselves relative to other solvents The preference of nonpolar molecules and groups for nonpolar environments is known as the hydrophobic interaction Hydrophobic Interactions The magnitude of the hydrophobic interaction is generally measured by the free energy of transfer AGtr of a nonpolar molecule in the gas liquid or solid state into water positive AGcr value indicates that the nonpolar molecule prefers a nonaqueous environment Transferring a solute molecule into a liquid involves 0 Creating a suitable cavity in the liquid 0 Introducing the solute molecule into the cavity 0 Rearranging the solute and liquid molecules to maximize favorable interactions between them The observed thermodynamics of this transition are the net effect of all these factors Interpretation is not always straightforward At room temperature the unfavorable transfer of a nonpolar molecule from a nonpolar liquid to water is primarily a result of the unfavorable change in entropy AHtr H 0 The unfavorable entropy change is thought to result from increased ordering of water molecules around the nonpolar group Surrounding water molecules appear to be more tightly packed than those of normal bulk water Water molecules cannot form hydrogen bonds with the nonpolar group Therefore they are generally believed to satisfy their hydrogen bond potential by forming a hydrogen bonded iceberg network among themselves at the nonpolar surface Hydrophobic Interactions and CP As the temperature is increased the ordered water shell around the nonpolar species tends to melt and become more like bulk water This melting process requires energy resulting in the observed large heat capacity CP A large CP is characteristic of aqueous solutions of nonpolar molecules and is generally proportional to the exposed nonpolar surface area of the solute The CP de nes the temperature dependence of both entropy and enthalpy The temperature dependence of the hydrophobic interaction can provide insights into its physical nature At TgtTH the entropy of transfer to water decreases but AHcr becomes unfavorable Ath the ASw becomes 0 Ts thought to be I40 C Large changes with temperature of AHcr and ASw mostly compensate and the value of AGcr changes much less than they do It is important to specify the measure of hydrophobicity being used AGw or Ktr At the high temperature Ts the persistence of low solubility of nonpolar groups in water is observed to be due to much weaker enthalpic interactions between the nonpolar species and water than between the nonpolar groups and the nonpolar solvent and between the water molecules in bulk water Hydrophobic Interactions to summarize Hydrophobic interactions do not result from repulsion between water molecules and nonpolar molecules and surfaces While favorable interactions do occur between water molecules and nonpolar molecules and surfacesthe magnitude of these interactions are less than the favorable van der Waals interactions in a nonpolar environment and the hydrogen bonding in liquid water Hydrophobic interaction results in nonpolar atoms molecules and groups to interact with each other rather than with water Intramolecular Interactions For molecules to interact with each other they must lose entropy which is energetically unfavorable The magnitude of the loss in entropy is dependent on the degrees of freedom that become xed as a result of the interaction In the case of intramolecular interactions the groups involved are incorporated within the same molecular scaffold which automatically limits the number of degrees of freedom by xing the relative distance and orientation of the groups involved Intramolecular and bimolecular interactions can be compared by means of the ratio of their equilibrium constants the effectlve concentratlon Kim A B A39B J Kinter A B 1 A39B Ki i effective concentration of A B Kinter Intramolecular Interactions The maximum effective concentration of two groups in an aqueous solution was believed to be but much greater values for effective concentration are usually observed Covalently linking the interacting moieties through a on network results in their concentration relative to each other to be much higher than would be possible were the two groups on separate molecules Interaction between the two groups results in the sacri ce of some fraction of the internal flexibility and conformational freedom of the molecule When there is no entropic difference between molecules with and without interaction between the groups their effective concentration is at its maximum value Interactions requiring proximity and orientation have very high maximum effective concentrations Interactions that are more tolerant and allow greater degrees of freedom have lower maximum effective concentrations Kimm A B A39B J Kinter A B 1 A39B K i effective concentration of A B Kinter Cooperativity of Multiple Interactions Multiple groups within a single molecule can behave differently from the same groups in solution The simultaneous presence of multiple interactions within a single molecule results in cooperativity between them Collectively these interactions can be much stronger than expected based on their individual strengt s Cooperatlvlty Is crltlcal for protelns Single interactions between groups within a polypeptide chain are not expected to be stable unless these groups lie in close proximity of each other within the covalent structure resulting in a high effective concentration Due to to the size and conformational flexibility of the unfolded protein groups attached to a moderate sized peptide have effective concentrations in the range of l0392l 0395 M depending on proximity Expected values for Kainquot observed equilibrium constant for individual hydrogen bonds salt bridges etc range from 4xl0393 to l0397 A KABlABlu 5 B B Unfolded Folded KobsU KABlABlu KAB association constant for free A and B ABu effective concentraoin oannd B in unfolded peptide Cooperativity of Multiple Interactions Multiple interactions among two or more pairs of groups within the same molecule often do not behave independently but assist or interfere with each other 5 In the example given if both interactions A B and C0D are B A possible simultaneously the interactions between one pair C D 2 43 y I YQJ of groups constrains the peptide increasing the effective concentration of the other pair I 9 E D B 7 This will proceed in a mutual manner with both interactions having the same effect on each other factor Coop is the degree of cooperativity between the interactions Unfolded Each interaction is more stable in the presence of the II 3 other than in its absence In polypeptides containing additional groups that interact NB CID simultaneously equilibria are exten e II I AIBu CIDu KnetKABABuKCDCDl KEFEFKGHGHIV The value of Kn is pathway independent The nal folded conformation is stableonly if the value of Knec is greater than unity Cooperativity of Multiple Interactions In considering a series of weak interactions the rst will be very weak with an equilibrium constant of I0393I 0397 A The rst interaction increases the effective concentration C39 B of the next pair of interacting groups resulting in a A V36 D V I C C39s D D SQgt Folded The net stabilities of conformations with additional weak KeefeD A interactions are even lower than that of the conformation B u lt y slightly larger equilibrium constant for the interaction by the factor Coop If the equilibrium constant of the second interaction is less than unity the product of the two equilibrium constants is lower than that of the rst 7 f Unfolded The process continues until the effective concentrations of additional interacting groups are suf ciently high to make the equilibrium constant for each additional interaction greater than unity with Kw increasing with each additional interaction AB CDI C with a single interaction 3 D II B In this manner a suf cient number of simultaneous weak ABu CDu COOP interactions can make the value of Knet greater than unity and provide a stable folded structure Intermediate partially folded structures with incomplete sets 0 wea interactions are unstable relative to the folded and unfolded states Mechanism of Protein Folding Folding Pathways 0 How does a protein fold into its native conformation 0 A protein cannot randomly explore all of the conformational possibilities until it achieves its native conformation Levinthal Paradox a l00 residue peptide sampling l0I3 conformations per second would take l085 sec to fold Universe is estimated to 20 billion years or 6xl0397 seconds old 0 Therefore proteins must employ an ordered pathway or set of pathways which ultimately allow the protein to achieve its native fold 0 There is the possibility that the observed folded state may not be the conformation with the lowest possible free energy but is the most stable of the kinetically accessible conformations 0 Proteins fold to a signi cant degree within l millisecond Monitoring protein folding requires techniques that allow rapid measurements of protein structure or degree of folding Circular dichroism fluorescence and pulsed HD exchange followed by NMR are some techniques used to follow protein folding Pulsed H D Exchange Pulsed HD exchange follows the folding of individual amino acids peptide bonds in a protein Peptide bond protons and other weakly acidic protons exchange with water NMR provides a means of following HD exchange Protein groups involved in hydrogen bonding within the protein exchange slowly In these experiments 3 A protein is denatured in the presence of D20 to fully exchange protons 3 It is then rapidly transferred to H20 at low pH pH 35 reducing hydrogen exchange 20 After a predetermined folding time the pH is increased and HD exchange is allowed Exchange is stopped after l040ps by rapidly lowering the pH again Folding is then allowed to continue 20 They HD ratio at each exchangeable site of the folded protein is then determined by NMR Klnetlc AnalySIS of Complex Reactions Kmeth of Unfoldlng mm mm W mm m m an a a Na mm mm mmm m mm quotWW 3 quotW WWW WWW WW and mm Wm mm W W m quota Wm Rm mm mm W W umlmngmdm Kmeth of Refoldmg mm 5 3 mm a W WWW mm mm mm mum Wm mm W m W55 m m 5 My mmquot aquot W 5 mm by um laldmg Sumng Wm m aMarmazlanal mm I vs an m mum m M zthhnum m m mm palyptpudt mnmm n mm mm hztwun m samus 5 mm 1 a Rdaldmgmzh absuvcz alslaw Wm hand lsamulzman an unfulde papulauan Wm m mm 0517376 lsamus quotmam glnually mm a n asmgl mu ansum m 5pm mm mmarmauanal hutmgznzny mm ur ald m M 5m Kinetic Analysis of Protein Folding Expulmznul appraathts In study pmum WW FAe AmW AnhumsEquauan mm mm m mm mm M m 5W km A e39 Kw kW uarzstuvu mak u passlhlz m allaw mm W nkw39mldenatl lrrsza lo mm l denat CD m H an m stalls mmmm nku lnk u mu Mmquot mum mm m mm mm mm mm an mm m mm m mm Wmme mm mm l m mg mm a a H wmuh dwlzhadudum2 all rrs k Mann me pm lts usual lallaw smglz txpanumal mm W m mung m dam prawdts ml and maxlmum zggm e332 W 04 w Far 3 twaszazz sysum kmw anal5x5 s rzlazwnly sum Harwan Plamng m mm agamsz dunk ylzlds lmur rzlazmnshlps m mm a chzvmn plat m horan m cm 5 Mm as m pm my and n m Wm handlm tq m m absuvu a dumzumm 5 an m o dumzumm Hierarchal Protein Folding 0 The folding process begins with the formation of marginally stable local orderstructure 0 These structure elements then interact locally to form intermediates of increasing complexity 0 Process continues ultimately yielding the native protein 0 Evidence supporting the premise of hierarchal protein folding 1 Many peptide fragments excised from proteins will assume their native conformation 1 Observed folding intermediates are consistent with a hierarchal folding process 2 Helix boundaries are xed by their primary sequence not so much by 3D interactions 1 Secondary structure can be predicted with reasonable accuracy even when longrange interactions are not accounted for or are suppressed 0 Sequence information de ning a speci c fold is both distributed throughout the polypeptide chain and is highly overdetermined 0 Initial folding events burst phase milliseconds 1 For many smallsingle domain proteins much of the secondary structure is establishe 1 Much of the driving force attributed to hydrophobic collapse hydrophobic groups coalesce and expel water 1 Initial collapsed state is molten globular 1 Side chains are extensively disordered 0 Intermediate folding events 5 I 000 milliseconds 1 Secondary structure stabilizes and nativelike tertiary structure appears 1 Side chains are still mobile 0 Final folding events S several seconds 1 Protein achieves native structure 1 Complex motions allow the protein to attain relatively rigid packing and hydrogen bonding 1 Remaining interior water molecules are expelled from the core Landscape Theory of Protein Folding Current Thinking Protein folding is envisioned to proceed on an energy surfacelandscape The landscape represents the conformational energy states available to a polypeptide Polypeptides fold via a series of conformational adjustments that reduce their free energy and entropy until the native folded state is achieved There is no single pathway or closely related set of pathways that a polypeptide must follow in achieving its native conformation Increasing Energy Suggests that landscape maw include local Decreasing Conforma ona energy minima and maxima therefore many Freedom possible transient folding intermediates may exist Folding of Multidomain and Multimeric Proteins 0 Large proteins may be composed of multiple domains or polypeptide chains Independent domains unfold and refold like singledomain proteins which can lead to complex unfolding curves for proteins in such cases domains may unfold under different conditions 0 Can also be varying degrees of interaction between the domain Interactions between domains can effect folding 0 Where the isolated domains are stable folding of the intact multidomain protein appears to occur by initial folding of the domains followed by association of the domains 0 Domain association is often the slowest step in the folding process domains may not be folded entirely correctly or because small adjustments are required for interaction between the domains 0 When association is slow step an intermediate can accumulate where domains are folded but impaired May lead to intermolecular interactions and precipitation Folding of Multidomain and Multimeric Proteins Largc mm m m campus a mum Mm up pnlypap da hams Fuidmg 039 uiigumnt mm m svmizr unmdamtmns Mann m puiypcptida m invuiwd Dom mama mm Mm Diigumarintmn mamas mm imam mm m puiypcptida mmpm Mummers 5 de am a m their Mai unlurmatiuns Mum uiigumnc mam m mm mummyu mi mqmr me menuMrs m 2 Dim enigmatici in warm mm c As m Mm the aim Mummers mm m up marmngc W mam Raterhmitmgsmp my be mm intmmiuuizrluidmg ummmm mm mm mm m m mgnmczntanargy barrier 7 Nu ganami mm an u an szi gamma a m m a is 2 mm siuw prams Flexibility of Protein Structure mm m a Pmth st 5 m m sum mu 5mm m w manta Ming dag unlumztmnzi mam Prmams an Mthuughtu39 axmmg m 2 r mum m mm mma mmru mewme me mamawart mm at tampamtum quotgm state warmth and Agk mums m mm m svmizr to than m mu miuuin mmm gt mm lm and mum ma rum DA 2 iungar mm said mgquot mm unlurmzmnzi mmm an mm On the iungcsttim saimtha aided aldermath is mgm 5mm and may tnnsmntiyszmpia m unluidad 5m mama57 5m m 039 rnidun at m pmth 5m an m swam unlurmtmnzi mam cm packing clamm m m pmth murmr g Conformational Motility Hydrogen Exchange Best evidence for extensive structural mobility is that internal groups in proteins react with appropriate reagents in solution buried groups either are occasionally at surface or reagent can permeate the protein Isotopic exchange with water H20 2H20 and 3H20 Hydrogen atoms covalently attached to various atoms exchange with solvent at different intrinsic rates depending on tendency of that atom to ionize Exchange of amide protons most often studied because these hydrogen atoms exchange on a useful time scale Rates of exchange impacted by temperature hydrogen bonding environment and degree of exposure Rate of exchange of individual H39s varies l00fold Protons involved in hydrogen bonding in the interior of Bsheets and OKhelices tend to exchange least readily Acid and base catalyzed exchange via transient protonation of CO and deprotonation of N H respectively Rates of exchange generally increase at elevated temperatures but in a complex manner Classical methods NMR and MS only provided insights into average number of protons exchanged Exchange of individual hydrogen atoms can be followed using 39HNMR Conformational Motility k1 k k folded T openigt Hexchanged K1 T1 1 1 Hydrogen exchange contlnued While proteins may sample unfolded state this is not likely responsible for the exchange of buried hydrogen atoms not all interior hydrogens exchange with same rate Local unfolding or breathing is often used to explain the exchange of interior hydrogen atoms The hypothetical open form is unstable and transient Under most conditions proteins demonstrate exchange rates consistent with kexltkI and rate KI x Alternative explanation involves rare instances of diffusion of solvent into the interior sites in the protein Supported by exchange in proteins in crystalline state Both require some degree of backbone conformational flexibility Available information suggests that different site in folded proteins likely exchange with solvent by a wide range of different processes depending on the protein and conditions
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