2008MSC'STRUCTURAL'BIOCHEMISTRY'

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1 LearningObjectives: 2008MSC'STRUCTURAL'BIOCHEMISTRY' Module'1' 'Amino'Acids'and'Proteins:'Structure'and'Function' 1 Understandthestructureandstereochemistryofalpha1aminoacids 1 Beabletodescribetheclassesofaminoacidsandtheirproperties 1 Beabletogiveexamplesofrareaminoacidsandmodifiedaminoacids 1 Beabletodescribeapeptide 1 Beabletodescribehowpolypeptidesareampholytes 1 Beabletoexplainthestructureofthepeptidebond 1 Describethestability&formationofthepeptidebond 1 Understandthatproteinsarepolypeptidesofadefinedsequence,foldedandbondedinaparticularway. Proteins: 1 Proteinsplayanenormousvarietyofroles: o Transportandstorageofsmallmolecules! Haemoglobin transportationofoxygen o Structuralframeworkofcellsandtissues! Alpha1Keratin structuralsupportinhair o Musclecontraction! ActionandMyosinFilaments o Immuneresponses! Immunoglobulins immuneresponse o BloodClotting! Fibrinogen bloodclotting o Enzymes! Procarboxypeptidase stomachenzyme 1 Structuresofthealpha1aminoacids o Tothea1carbonofeveryaminoacid,thereis:! AnaminogroupNH3)! AcarboxylicacidgroupCOOH)! Ahydrogenatom! AsidechainRGroup) o Differenta1aminoacidsaredistinguishedbytheirsidechains. 1 ZwitterionFormation: 1 Structuresofthea1aminoacids:! Thereare20differentkindsofaminoacidsthatarecommonlyincorporatedintoproteins.! TheaminoacidsareclassifiedbytheirsidechainsRGroups)

2 1 KEYPOINTS CHAPTER5! Alpha amino acids have a chiral central alpha carbon to which an alpha amino group, a carboxylicacidgroup,ahydrogenandavariabler1groupareattached.! Aminoacidshaveacidicandbasicpropertiesduetotheiraminoandcarboxylicacidgroups.! The'pKa'of'the'COOH'is'around'2'and'the'pKa'of'the'amino'group'is'around'10! AtphysiologicalpH,aminoacidsarefoundasazwitterion,withanionisedcarboxylicacid COO1)andapositivelychargedaminogroupNH3+)! AminoacidshavedifferentchargesatdifferentpHvalues.! Thereare20commonaminoacidsthatdifferintheirside1chains. Some rare amino acids includeselenocysteineandpyrrolysine KNOWONEOFTHESEASANEXAMPLE.! Allalphaaminoacidsexceptglycinearechiralandin'nature'the'LIamino'acids'dominate.! Theside1chainsofthealphaaminoacidscanbeclassifiedas: Aliphaticopenchain) HydroxylorSulphurcontaining Aromatic Basic Acidicortheiramidecounterparts! AromaticaminoacidshaveaUVspectrumthatcanbeexploitedtostudyproteins! Someaminoacidsundergoposttranslationalmodification.! Alpha amino acids can covalently link together to form an amide bond this is called a peptidebond.! Amino' acid' polymers' are' called' polypeptides' ' they' possess' an' NIterminus' free' ended' amide' group)' and' a' CIterminus' free' ended' carboxylic' acid' group).' Polypeptides' are' written'from'their'niterminus'to'the'citerminus.''! Polypeptidesthatentailanymorethan50aminoacidsareclassifiedasproteins.'! Polypeptides exhibit Polyampholytic behaviour the ability to bind or detach protons/h atomsatmultiplelocationsdependentupondifferentphlevels.'! Theamideorpeptidebondhasplanarcharacteristicsandrotationaroundthealpha1carbon toalpha1amidebond itisveryrestrictedandhasahighdegreeofdoublebondcharacter.'! Peptide bonds are mainly in the trans form except proline which CAN adopt the cis conformation stilldoesn tlikeitthough).'! Theaminoacidsequenceofaproteiniscalleditsprimarystructure.'! Proteins come with many different protein sequences and molecular weights all with varyingcharacteristics.'! Conjugated proteins have other chemical groups associated with them in addition to a polypeptidechainprostheticgroupsattachedalso).' PART'2 1 LearningObjectives! Beabletodescribethefeaturesofregularpolypeptidestructures! Explain what secondary structure is and be able to describe helices and beta sheets, also Ramachandran plots the graph thing with the different angles at which the bonds can rotate.! Describethestructuralfeaturesoffibrousproteinsincludingkeratins,fibroin,collagenand elastin.! Understandthatdifferentfoldsresultindifferentfunctionsandwhattertiarystructureis.! Understandinformationforfoldingandthethermodynamicsoffolding.! Understandindetailtheroleofdisulphidebonds! Describethekineticsofproteinfolding.! Understandtheroleofchaperones! Understandthatproteinmisfoldingcausesdiseasemadcowdiseaseforexample).! Beabletoprovideexamplesofproteintoproteininteractionsandquaternarystructures.

3 o! Thehydrophilicresiduesred)areonthesolvent1exposedouter)surfaceoftheprotein they rehydrophilic. Mostoftheinformationfordeterminingthe3Dstructureofaproteiniscarriedintheaminoacid sequenceofthatprotein! Under harsh conditions, a protein loses its functional 3D structure/configuration this processiscalleddenaturation! Denaturingconditionsinclude: Increasedtemperature=Hydrogenbondsruined AciditypH)=Ionicandhydrogenbondsruined OrganicSolvents/Urea=Destructionofhydrophobicinteractions! Example thedenaturationandrefoldingofribonucleasea: o Thestabilityofthefoldedstructureofaglobularproteindependsontheinterplayofthreefactors:! Theunfavourableconformationalentropychange,whichfavourstheunfoldedstate! Thefavourableenthalpycontributionarisingfromintramolecularinteractions! Thefavourableentropychangearisingfromtheburyingofhydrophobicgroupswithinthe molecule o TheroleofDisulphidebonds:! Somefoldedproteinsarestabilisedbyinternaldisulphidebonds,inadditiontonon1covalent forces.! Disulfidebondsarerelativelyrareandarefoundprimarilyinproteinsthatareexportedfrom cells,suchasribonuclease,bptiandinsulin.! One explanation is that the environment inside most cells is reducing and tends to keep sulphur1hydrolgroupsinthereducedstate.! Externalenvironments,forthemostpart,areoxidisingandstabilisebridges. 1 KineticsofProteinFolding:! Thefoldingofglobularproteinsfromtheirdenaturedconformationsisaremarkablyrapid process,oftencompleteinlessthanasecond! Proteinfoldingisnotacompletelyrandomsearchthroughavastconformationalspace.! Rapidkineticstudiesshowthatfoldingtakesplacethroughaseriesofintermediatesteps! Foldingcanbedelayedbytrappingofmoleculesin off1pathway states.! Oneofthemostcommonoffoldingerrorsresultsfromtheincorrectcis1transisomerisation oftheamidebondadjacenttoaprolineresidue shownbelow: o! Proteinfoldingandassemblyinvivoissometimesaidedbychaperoneproteins. Proteinfoldingandaggregation:! Apathwaymodelshowingvariousproteinconformationsandtheirinterconversions! Thesameinformationshowninanenergylandscapemodelbelow)

4 1 QuaternaryStructureofProteins: o Multi1subunitProteins:Homotypicproteintoproteininteractions:! Theinteractionsbetweenthefoldedpolypeptidechainsinmulti1subunitproteinsareofthe samekindthatstabilisethetertiarystructure: Saltbridges HydrogenBonding VanDerWaalsForceshydrophobicinteractions) DisulfideBonding! Theseinteractionsprovidetheenergytostabilisethemulti1subunitstructure.! Associationofpolypeptidechainstoformspecificmulti1subunitstructuresisthequaternary levelofproteinorganisation. 1 KeyConcepts:! Polypeptides chains can fold into regular patterns that are called secondary structures alphahelixandbetasheets.! Thebasisfortheseregularfoldingpatternsisthepeptidebonds althoughtheamidebond hasrestrictedrotation,thenh1calpha)orphiø)andcalpha)1coohorpsiψ)bondscan adoptcertainangles butcanneverbothbe0degreesstericinterference)! HydrogenbondingbetweentheNHandCOOHofdifferentaminoacidsinthepolypeptide chain is the basis of the stabilisation of the structure of alpha1helices and beta1sheets 4 residuesapart).! In proteins, the right handed alpha helix predominates, with 3.6 residues per turn, and a pitchof0.54nm.thecarbonylonresiduei$ishydrogenbondedtotheaminoprotonthatis4 residuesawayinthedirectionofthec1terminusi+4) Thesidechainspointoutwardsfromthehelix.! InBetaSheets,therearetwoormorestandsofpolypeptidethatcanbequitedistantaway intheprimarysequence.twostrandscanbearrangedantiparallelorparallel,dependingon theorientationoftheterminus. When antiparallel, the N1terminus and C1terminus are arranged in opposite directions,causingthehydrogenbondsbetweentheaminoandcarbonylgroupsto belinear.sidechainsareaboveorbelowtheplaneofthesheet. Whenparallel,thestrandsarearrangedsothattheN1terminusandC1terminusrun in the same direction. In this case, the hydrogen bonds between the amino and carboxylgroupsarenotlinear.sidechainswillbeaboveorbelowtheplaneofthe sheet.! Stericinteractionsdeterminepeptideconformation.Allowedanglesofphiandpsiforthe differenttypesofsecondarystructurearerepresentedinaramachandranplot.! Fibrous proteins play structural roles and have generally only one type of secondary structureandtheiraminoacidcompositionreflectstheirfunction.! Keratinispredominantlyalpha1helicalinstructureandhasacoiled1coilarrangement.They are initially coiled right1handedly, and then coiled together in a left1handed manner. The SerineandAlaninesidechainsinteractandfitsnuglytogetherinalpha1keratin.! Collagenisatriplehelixofthreepolypeptidechainsthatarelefthandedhelices,theseare thenwrappedaroundeachotherinaright1handedsense withhydrogenbondingbetween thechainsandeverythirdresidueisglycineinthesequence. Hydroxyproline and hydroxylysine are present Hydroxyproline is essential for stabilising collagen, and vitamin c ascorbic acid) assists in the production of hydroxyprolinefromproline areactioncatalysedbypropylhydroxylase. Thereiscovalentbondsbetweenthecollagenchainsandthedegreeofcrosslinking increases with age = skin becomes more brittle with age as the crosslinking increases.! Elastinhaslittlesecondarystructureandcontainslysineresiduesthatcrosslink.

5 1 KeyPoints! These sticky spots cause deoxy1haemoglobin S molecules to associate cling together) to formthelongfibrousaggregates.! SickleCellAnaemiaRBC sstilltransportoxygen,butatamuchlowerrateofefficiencydueto theirdeformed,elongatedshape.! Thesebloodcellshavedifficultypassingthroughcapillaries thiscausesorgandamagedue toreducedcirculation.! Oxygenhasalowsolubilityanddiffusesslowlyinplasma,haemoglobinandmyoglobinare moleculesinoxygentransportandstoragetoproviderespiringtissueswithasteadysupply ofoxygen.! Myoglobinhasonepolypeptidechainandatertiaryfold/structure producingoneoxygen bindingsite.! Haemoglobin has 4 polypeptide chains 2 alpha and 2 beta) joined together into a quaternarystructuremainlythroughtheuseofsaltbridges).eachsubunithasanoxygen binding site a heme ring/group), so in total one haemoglobin molecule binds 4 O 2 molecules.! The oxygen binding site in myoglobin and haemoglobin is a heme prosthetic group. The hemeiscomposedofaprotoporphyrinringwith4pyrrolenitrogensthatcoordinatetoa centralfeii. Theoxygencoordinateddirectlytotheiron.The6 th coordinationpositionofironis fromthenitrogenofahistadineresiduecalledtheproximalhistadine)completing theoctahedralgeometryaroundtheironatom4xnbondstosurroundingamino groups,onebondtoo 2 andonebondtohistadine).! MyoglobinandhaemoglobinpreventtheFe 2+ frombeingoxidisedtofe 3+ whichcan tcarry oxygen).! We can analyse the binding of oxygen to myoglobin or haemoglobin by use of a binding curve where the number of myoglobin or haemoglobin molecules carrying oxygen saturation)isplottedagainstthepatrialpressurepresentofoxygentheppofoxygeninthe lungsisaround100mmhg,andinthetissuesisaround30mmhg).! MyoglobinhasalowP 50 valueandisastrongbinderofoxygenthep 50 valueiswherethe saturation of oxygen is 50%). Myoglobin is therefore a storage molecule for oxygen understandwhythisisso.theshapeofthemyoglobinoxygenbindingcurveishyperbolic.! Haemoglobinisanoxygentransportprotein.ItmustpickupoxygeninthelungswherePPof oxygenishigh100mmhg)anddeposititinthetissueswhereppofoxygenislow30mmhg). Thequaternarystructureofhaemoglobinallowscooperativebindingofoxygen.Theshape or conformation of haemoglobin subunits changes as oxygen binds ALLOSTERIC EFFECT). Thisconformationchangeistransmittedacrosstheproteinfromhemeringtohemering.! Oxygen is an allosteric effector of haemoglobin. When oxygen binds to one haemoglobin subunitorhemegroup),theaffinityofoxygenattheremainingun1boundsitesisincreased, makingoxygenmorelikelytobindtothehbmolecule. Onceasecondoxygenbinds,thisfurtherincreasedtheaffinityandlikelihoodthat anotheroxygenwillbindetc.thisisbecauseoftheconformationalchangestothe quaternary structure of haemoglobin that results in a change of function T1state changestor1state).! The same process happens in reverse once one oxygen is released, the conformational changesinthatsubunitencouragetheremovalofoxygenformtheothergroupsdecreasing thehaemoglobinsaffinityforoxygen.thismakesitmuchmorelikelythatasecondsubunit willloseitsoxygenandrearrangeitsconformation,thereforeencouragingfurtherrelease etc. Because of this allosteric effect, the oxygen binding curve of haemoglobin is SIGMOIDALinshape.! Inthefullyoxygenatedstate,haemoglobinisreferredtobeingintheR1State.Inthefully deoxygenatedstate,haemoglobinisreferredtobeinginthet1state.

6 Key salt1bridges between subunits are disrupted when haemoglobin changes its quaternarystructure/conformationinthechangefromthet1statetor1state.! Cooperative binding and transport of oxygen are only part of the allosteric behaviour of haemoglobin.theby1productofrespirationcarbondioxide)diffusesfromthemitochondria withinthecellsandoutintothetissues. ThecarbondioxideinteractswithwaterandisconvertedtoH + ionsandbicarbonate thebohreffect acceleratedthroughtheuseofcarbonicanhydrase),thisresultsin thetissuesbecomingmuchmoreacidicloweredph). Haemoglobin,wheninthepresenceoflowpHorhighacidity,favourstheT1state oxygenremoval/release).! A small proportion of the bicarbonate produced reacts with the N1terminus group of the haemoglobinpolypeptidechaintoformacarbamateswhichisthentransportedbacktothe lungswiththeactualhaemoglobinmoleculebicarbonate'does'not'bond'to'the'heme' GROUP). Hydrogen ions released in this action further increase the acidity and therefore BohrEffect. AdecreaseinpHleadstoastabilisationinthedeoxystateofhaemoglobinT1state)! Bisphosphoglycerate 2,31BPG) also lowers the affinity of Haemoglobin for oxygen AN ALLOSTERIC EFFECTOR just like oxygen itself). 2,31BPG binds in the central cavity of haemoglobin.! Sickle Cell Anaemia is a genetic disease that results in a mutant, deformed haemoglobin molecules. A surface glutamic acid negatively charged at physiological ph) is replaced by Valine a hydrophobic, neutrally charged Amino acid. This hydrophobic AA causes a hydrophobicpatchtobecomeexposedonthesurfaceofthehaemoglobinmoleculethey clumptogether,tryingtogetawayfromtheaqueoussolution). Thesehydrophobicpatchesondifferenthaemoglobinmoleculesclumptogetherto avoidtheaqueousenvironment,causinglongfibrousaggregatestoform. Thebloodcellsbecomedeformedandhavedifficultypassingthroughcapillaries. PART'4 ContractileProteinsandMolecularMotors 1 LearningObjectives! Understandactinandmyosin! Beabletoexplain,insimpleterms,thestructureofthemuscle! Understandthemechanismofcontraction theslidingfilamentmodel! Beabletoexplaintheregulationofcontractionandtheroleofcalcium 1 MusclesandOtherActin1MyosinContractilesystems! Themechanicalworkofmotioniscarriedoutbymotorproteins! This motion may involve the whole organism, parts thereof, cells, or subcellular constituents.! Proteinconformationalchangeismediatedbythebinding,hydrolysisandreleaseofATP allkeyfeaturesofmotorproteinfunction.! Allmuscles,aswellassomeothercontractilesystems,arebasedontheinteractionoftwo majorproteins actinandmyosin Thesearereferredtoastheactin1myosincontractilesystems.! Microtubulesarefilamentousstructuresmadeoftheproteintubulin Theirfunctionsinclude: o Beatingofciliaandflagella o Movementofchromosomesandorganelles.! Thebiologicalsystemsthatproducemovementshareonecommonfeature allhydrolyse ATP.