Protein folding

Preface

Structuredeterminesfunction.Justknowingthegenomesequencedoesnotenableustofullyunderstandthefunctionofaprotein,letalonehowitworks.Proteinscanassemblethemselvesinthecellularenvironment(specificpH,temperature,etc.)byvirtueoftheirinteractions.Thisself-assemblyprocessiscalledproteinfolding.

Theproblemofproteinfoldingislistedasanimportanttopicof"Biophysicsinthe21stCentury".ItisamajorbiologicalproblemthathasnotyetbeenresolvedbytheCentralPrinciplesofMolecularBiology.Predictingthetertiarystructureofaproteinmoleculefromtheprimarysequenceandfurtherpredictingitsfunctionisanextremelychallengingtask.Thestudyofproteinfolding,especiallytheearlystagesoffolding,thatis,thefoldingprocessofnascentpeptidesisafundamentalissuethatfinallyclarifiesthecentralprinciple.Inthisfield,newdiscoveriesinrecentyearshavediscoveredthetraditionofspontaneousfoldingofnascentpeptides.Theconcepthasbeenfundamentallyrevised.Amongthem,X-raycrystaldiffraction,variousspectroscopictechniques,andelectronmicroscopytechniqueshaveplayedanextremelyimportantrole.Atthe13thInternationalConferenceonBiophysics,NobelPrizewinnerErnstemphasizedinhisreportthatoneofthemainadvantagesofNMRforstudyingproteinsisthatitcanstudythedynamicsofproteinmoleculesingreatdetail,thatis,thedynamicstructureorstructure.Therelationshipbetweenexerciseandthefunctionofproteinmolecules.ThecurrentNMRtechnologyhasbeenabletoobservethemovementofproteinstructuresinthetimedomainfromsecondstopicoseconds,includingthemovementofthemainchainandsidechains,aswellasthefoldingandunfoldingprocessesofproteinsundervarioustemperaturesandpressures.Thestructuralanalysisofproteinmacromoleculesisnotonlytosolveaspecificstructure,buttopaymoreattentiontothefluctuationandmovementofthestructure.Forexample,enzymesandproteinsthattransportsmallmoleculesusuallyhavetwoconformations,ligand-boundandunligand-bound.Structuralfluctuationwithinaconformationisanecessarypreludetoconformationaltransformation.Therefore,itisnecessarytocombinespectroscopy,spectroscopyandX-raystructureanalysistostudythebalanceofstructuralfluctuations,conformationalchanges,andvariousintermediatestatesformedduringthechange.Foranotherexample,inordertounderstandhowaproteinfolds,itisnecessarytoknowthetimescaleandmechanismofseveralbasicprocessesduringfolding,includingtheformationofsecondarystructures(helixandfolding),coiling,long-rangeinteractions,andunfoldedpeptides.Totalcollapse.Avarietyoftechniquesareusedtostudysub-processes,suchasfastnuclearmagneticresonance,fastspectroscopytechniques(fluorescence,far-ultravioletandnear-ultravioletcirculardichroism).

Researchoverview

Inorganisms,theflowofbiologicalinformationcanbedividedintotwoparts:ThefirstpartisthetransferofgeneticinformationstoredinDNAsequencesintoproteinsthroughtranscriptionandtranslation.Intheprimarysequence,thisisthetransmissionofone-dimensionalinformation,andthetripletcodonmediatesthistransmissionprocess;thesecondpartisthenaturalproteinformationofthepeptidechainthroughthefoldingprocessofhydrophobiccollapse,spatialtwisting,andsidechainaggregation.Conformation,whileobtainingbiologicalactivity,soastoexpresslifeinformation;andproteinasanexpressionvectorforlifeinformation,thespecificspatialstructureformedbyitsfoldingisthebasisforitsbiologicalfunction,thatis,thisone-dimensionalinformationisdirectedtothree-dimensionalTheprocessofinformationtransformationisnecessaryfortheexpressionofvitality.

Sincethe1960s,AnfinsenhasbasedontheexperimentalresultsofreducingdenaturationofbovinepancreasRNasewithoutanyhelpbyremovingthedenaturantandreducingagenttorestoreitsnaturalstructure.Sincethe"self-assemblytheory"of"theaminoacidsequenceofapolypeptidechaincontainsalltheinformationnecessarytoformathermodynamicallystablenaturalconformation",withtheextensivedevelopmentofproteinfoldingresearch,peoplehavefurthersupplementedtheproteinfoldingtheoryAndexpansion.Anfinsen's"self-assemblythermodynamichypothesis"hasbeenprovedbymanyinvitroexperiments.Indeed,manyproteinscanundergoreversibledenaturationandrenaturationinvitro,especiallysomesmallmolecularweightproteins,butnotallproteinsarethesame.Andbecauseofspecialenvironmentalfactors,thefoldingofproteinsinthebodyisfarfromthecase.

ThefoldingofproteinsinthebodyoftenrequirestheparticipationofothercofactorsandisaccompaniedbythehydrolysisofATP.Therefore,in1987,Ellisproposedthe"assistedassemblytheory"ofproteinfolding.Thisshowsthatproteinfoldingisnotonlyathermodynamicprocess,itisobviouslyalsocontrolledbykinetics.Somescholarsbasedonthephenomenonthatsomeproteinswithsimilaraminoacidsequenceshavedifferentfoldingstructures,whileotherproteinswithdifferentaminoacidsequencesarestructurallysimilar.TheyproposedthehypothesisthatmRNAsecondarystructuremaybeusedasageneticcodetoinfluenceproteinstructure..Butsofar,thereisnoexperimentalevidenceforthishypothesis,onlysomepuremathematicalarguments[3].So,howexactlydoestheaminoacidsequenceofaproteindetermineitsspatialconformation?Researchershavedonealotofexcellentworkonthisissue,butsofarourunderstandingoftheproteinfoldingmechanismisstillincomplete,andthereareevenerroneousviewsinsomeaspects.

AtypicalresearchexamplethathasmadeimportantcontributionsinthisregardisthestudyofthedenaturationandrenaturationofbovinepancreaticribonucleasebytheC.B.AnfinsongroupintheUnitedStates.Bovinepancreaticribonucleasecontains124aminoacidresiduesandiscomposedof8sulfhydrylgroupstoform4pairsofdisulfidebonds.Itcanbecalculatedthatthereare105possiblewaysforthe8sulfhydrylgroupsintheenzymemoleculetoform4pairsofdisulfidebonds,whichprovidesaquantitativeestimationindexforrefoldingrecombination.Undermildalkalineconditions,8molesofconcentratedureaandalargeamountofmercaptoethanolcancompletelyreducethefourpairsofdisulfidebonds,thewholemoleculebecomesirregularlycoiled,andtheenzymemoleculeisdenatured.Dialysisremovesurea.Inthepresenceofoxygen,thedisulfidebondisre-formedandtheenzymemoleculeiscompletelyrenatured.Thepairedsulfhydrylgroupsinthedisulfidebondarethesameasnatural.TherenaturedmoleculecanbecrystallizedandhasthesameX-raysasthenaturalenzymecrystal.Diffractionpatternsconfirmthattheenzymemoleculenotonlyrefoldsspontaneouslyduringtherenaturationprocess,butalsoonlyselectsoneofthe105possibledisulfidebondpairingmodes.

Theoreticalmodel

Framemodel

(FrameworkModel)

Frameworkmodel[4]AssumingthatthelocalconformationoftheproteindependsonthelocalAminoacidsequence.Intheinitialstageofthefoldingprocessofthepolypeptidechain,unstablesecondarystructureunitsareformedrapidly;called"flickeringclusters",andthenthesesecondarystructurescomeclosetocontacttoformastablesecondarystructureframework;finally,thesecondarystructureTheframesarespliced​​together,andthepeptidechainsaregraduallytightened,formingthetertiarystructureoftheprotein.Thismodelbelievesthatevenasmallmoleculeproteincanbefoldedpartbypart,andthesubdomainformedduringitisanimportantstructureofthefoldingintermediate.

Collapsemodel

(HydrophobicCollapseModel)

Inthehydrophobiccollapsemodel[5],thehydrophobicforceisconsideredtobeintheprocessofproteinfoldingThedecisiveforcefactor.Beforeanysecondarystructureandtertiarystructureareformed,arapidnon-specifichydrophobiccollapseoccursfirst.

Adhesionmechanism

(Diffusion-Collision-AdhesionModel)

Thismodelbelievesthatthefoldingofaproteinstartsatseveralsitesonthestretchedpeptidechain,Theformationofunstablesecondarystructureunitsorhydrophobicclustersatthesesitesmainlydependsontheprogressofthelocalsequenceormid-range(3-4residues)interactiontomaintain.Theyspread,collide,andadheretoeachotherinanon-specificBrownianmotion,leadingtotheformationoflargestructuresandthusincreasingstability.Furthercollisionsformasphericalstructureofamoltensphericalintermediatewithahydrophobiccoreandasecondarystructure.Thesphericalintermediateisadjustedtoadense,inactive,highlyorderedmoltensphericalstructuresimilartothenaturalstructure.Finally,theinactive,highlyorderedmoltensphericalstateistransformedintoacompleteandvigorousnaturalstate.

Growthmodel

(Nuclear-Condensation-GrowthModel)

Accordingtothismodel,acertainregioninthepeptidechaincanforma"foldednucleus"Withthemasthecore,theentirepeptidechaincontinuestofoldtoobtainanaturalconformation.Theso-called"crystalnucleus"isactuallyanetworkstructureformedbysomespecialaminoacidresidues,whichissimilartothenaturalinteraction.Theseresiduesarenotmaintainedbynon-specifichydrophobicinteractions,butbyspecificinteractions.Theresiduesformedatightpacking.Theformationofcrystalnucleiistherate-limitingstepintheinitialstageoffolding.

Impositionmodel

(Jig-SawPuzzleModel)

Thecentralideaof​​thismodel[9]isthatthepolypeptidechaincanbefoldedinmanydifferentwaysIntheprocessoffoldingalongeachpath,therearemoreandmorenaturalstructures,andfinallyanaturalconformationcanbeformed.Moreover,thefoldingspeedalongeachpathisfaster.Comparedwiththesingle-pathfoldingmethod,thepolypeptidechainspeedisfaster.Fast,ontheotherhand,smallchangesormutationsintheexternalphysiologicalandbiochemicalenvironmentmayhaveagreaterimpactonasinglefoldingpathway.Forfoldingmethodswithmultiplepathways,thesechangesmaygiverisetoacertainfoldingpathway.Itdoesnotaffectotherfoldingpathways,andthereforewillnotinterferewiththefoldingofthepolypeptidechainasawhole,unlessthechangescausedbythesefactorsaretoolargetofundamentallyaffectthefoldingofthepolypeptidechain.

Latticemodel

Latticemodel(alsoreferredtoasHPmodel)wasfirstproposedbyDilletal.in1989.Thegridmodelcanbedividedintotwotypes:two-dimensionalmodelandthree-dimensionalmodel.Thetwo-dimensionalgridpointmodelistogenerateorthogonalunit-lengthgridsinaplanespace,andeachaminoacidmoleculeisplacedontheintersectionofthesegridsintheorderofthesequence,andtheadjacentaminoacidmoleculesinthesequenceWhenplacedinthegridpoint,itmustbeadjacent,thatis,thedistancebetweenadjacentaminoacidmoleculesinthegridpointmodelis1.Butitshouldbenotedthateachintersectioninthegridcanonlyplaceoneaminoacidmoleculeatmost.Ifanaminoacidmoleculeinthesequencehasbeenplacedatthisposition,thesubsequentaminoacidmoleculecannotbeplacedatthisposition.Gridpoint.Ifduringtheprocessofplacingtheaminoacidmolecule,itappearsthattheaminoacidmoleculecurrentlytobeplacedhasnopositiontobeplaced,itmeansthattheconfigurationisunreasonableandneedstoberepositioned.Thethree-dimensionalgridpointmodelissimilartothetwo-dimensionalgridpointmodel.Itisathree-dimensionalgridofunitlengthgeneratedinathree-dimensionalspace.Themethodofplacingaminoacidmoleculesinthegridisthesameasthatofthetwo-dimensional,butwhenplacingaminoacidmoleculesinthetwo-dimensionalgridmodel,exceptforthefirsttwoaminoacidmoleculesinthesequence,onlythreedirectionscanbeselected,whichiscomplicatedinthethree-dimensionalgridmodel.Thedegreehasincreasedalot,andthereareuptofivedirectionsforplacingaminoacidmolecules.

Molecularchaperones

In1978,LaskeydiscoveredinvitrophysiologicalionicstrengthexperimentsofhistonesandDNA,andfoundthattheremustbeakindofacidicproteininthenucleus---nuclideWhen(nucleoplasmin)exists,thetwocanassembleintonucleosomes,otherwiseprecipitationwilloccur.Basedonthis,Laskeycallsita"molecularchaperone."Molecularchaperonereferstotheonethatcanbindandstabilizetheunstableconformationofanotherprotein,andcanpromotethefoldingofnewpolypeptidechains,theassemblyordegradationofpolymers,andthetransmembranetransportoforganelleproteinsthroughcontrolledbindingandrelease.Protein-like[10,11].Molecularchaperonesaredefinedintermsoffunction.Allproteinswiththisfunctionaremolecularchaperones,andtheirstructurescanbecompletelydifferent.Thisconcepthasnowbeenextendedtomanyproteins,andthemolecularchaperonesthathavebeenidentifiedmainlybelongtothreetypesofhighlyconservedproteinfamilies[12]:stress90family,stress70family,andstress60family.Amongthem,thestress60familyexistsinthemitochondriaofeukaryotes(calledHsp58inmammals)andchloroplasts(calledcpn60).Inthecytoplasmofprokaryotes,itiscalledGroEL.

Significance

Theclarificationoftheproteinfoldingmechanismwillrevealthesecondsetofgeneticcodesinlife,whichisitstheoreticalsignificance.Thestudyofproteinfolding,inanarrowersense,istostudytheformationofaspecificthree-dimensionalstructureofaprotein,itsstability,anditsrelationshipwithitsbiologicalactivity.Conceptually,therearethermodynamicandkineticproblems;proteinfoldinginvitroandintracellularfolding;andtheoreticalresearchandexperimentalresearch.Themostfundamentalscientificquestionhereishowdoestheprimarystructureofthepolypeptidechaindetermineitsspatialstructure?Sincetheformerdeterminesthelatter,theremustbeacertaindefiniterelationshipbetweentheprimarystructureandthespatialstructure.Doesthisalsohaveasetofcodeslikenucleotidesthatdeterminethesequenceofaminoacidsthroughthe"triplecode"?Somepeoplecallthisimaginarycodethatdeterminesthespatialstructureoftheprimarystructureasthe"secondgeneticcode."

Ifthe"triplecode"hasbeendecipheredbuthasactuallybecomeaclearcode,thendecipheringthe"secondgeneticcode"isexactlythe"proteinstructureprediction"thattheoreticallysolvestheproteinfoldingproblemmostdirectlyThisisoneofthelastfewmysteriesofproteinresearchthathavenotyetbeenrevealed."Proteinstructureprediction"isatheoreticalthermodynamicproblem.ItistopredictthespecificspatialstructuredeterminedbytheAnfinsenprinciplebasedonthemeasuredprimarysequenceoftheprotein.Thedeterminationofproteinaminoacidsequence,especiallythenucleotidesequenceencodingproteinhasalmostbecomearoutinetechnique.FromcomplementaryDNA(cDNA)sequence,aminoacidsequencecanbededucedaccordingtothe"triplecode".Thesemolecularorganismshavemademajorbreakthroughsinthelastcentury.Learningtechnologyhasgreatlyacceleratedthedeterminationofproteinprimarystructure.Atpresent,thereareabout170,000proteinprimarystructuresintheproteindatabase,butonlyabout12,000proteinswhosespatialstructurehasbeendetermined.Manyoftheseproteinsareverysimilarhomologousproteins,whiletheonlytrulydifferentproteinsareMorethan1,000.WiththesuccessfulcompletionoftheHumanGenomeProjectandtheinterpretationoftheentiresequenceofhumanDNA,thedatagrowthoftheproteinprimarystructurewillinevitablyexplode,andthespeedofspatialstructuredeterminationisfarbehind,sotherewillbemorechangesbetweenthetwo.Largedistancesrequiremorepredictionofproteinstructure.

Prospects

Atthesametime,italsohasimportantpotentialapplications,suchasthefollowingaspects:

Inclusionbodyrenaturation

▲TheuseofDNArecombinationtechnologycanintroduceforeigngenesintohostcells.However,theexpressionproductsofrecombinantgenesoftenforminactiveandinsolubleinclusionbodies.Theclarificationofthefoldingmechanismwillbeofgreathelptotherenaturationofinclusionbodies.

Protein

▲ThedevelopmentofDNArecombinationandpeptidesynthesistechnologyenablesustodesignlongerpolypeptidechainsaccordingtoourwishes.Butbecausewecannotknowwhatconformationthispolypeptidewillfoldinto,wecannotdesigntheproteinweneedandhaveaspecificfunctionaccordingtoourownwishes.

Pathogenicmechanism

▲Manydiseases,suchasAlzheimer's,MadCow(BSE),TransmissibleSpongiformEncephalopathy(CJD),MuscleAtrophiclateralsclerosis(ALS)andParkinson'sdisease(Parkinson's)arecausedbymutationsinsomeimportantproteinsincells,leadingtoproteinaggregationormisfolding.Therefore,anin-depthunderstandingoftherelationshipbetweenproteinfoldingandmisfoldingwillbeofgreathelptotheelucidationofthepathogenicmechanismofthesediseasesandthesearchfortreatmentmethods.

Revealingfunction

▲Thedevelopmentofgenomesequencehasenabledustoobtainalargenumberofproteinsequences.Theacquisitionofstructuralinformationisveryimportantforrevealingtheirbiologicalfunctions.Relyingontheexistingmethods(X-raycrystaldiffraction,NMRandelectronmicroscopy)todeterminethestructureoftheproteinrequiresalongtime,sothepaceofstructuralanalysishaslaggedbehindthepaceofdiscoveringnewproteins.Althoughthestructurepredictionmethodisfast,itisnotreliable.Onlywhenwehaveabetterunderstandingofthemaintenanceofproteinstructureandthephysicalandchemicalfactorsthatdriveproteinfolding,canthismethodbefundamentallyimproved.Inaddition,ourresearchontherelationshipbetweenstructureandfunction,suchasproteininteraction,theroleofligandsandproteins,alsodependsontheelucidationoftheproteinfoldingmechanism.

Foldingdisease

Theaminoacidsequenceoftheproteinmoleculedoesnotchange,butchangesinitsstructureorconformationcanalsocausedisease,whichiscalled"conformationaldisease"or"folding"sick".

MadcowdiseaseiscausedbytheinfectionofPrionprotein,whichcanalsoinfectpeopleandcauseneurologicaldiseases.Inanormalbody,Prionisaproteinrequiredfornormalneuralactivity,whiletheprimarystructureofdisease-causingPrionandnormalPrionisexactlythesame,butthespatialstructureisdifferent.

DiseasecausedbymolecularaggregationorevenprecipitationorfailuretobetransportedinplaceduetoabnormalproteinfoldingincludeAlzheimer’sdisease,cysticfibrosis,familialhypercholesterolemia,familialamyloidosis,Certaintumors,cataracts,etc.

Becauseofthevitalroleofmolecularchaperonesinproteinfolding,mutationsinthemolecularchaperoneitselfwillobviouslycauseabnormalproteinfoldingandcausefoldingdisease.Withthedeepeningofproteinfoldingresearch,scientistswilldiscoverthetruecausesofmorediseasesandmoretargetedtreatments,anddesignmoreeffectivedrugs.