StructuralBioinformatics
Lecture1XavierDaura
Institut deBiotecnologia i deBiomedicina
Module2:CoreBioinformatics
MScinBioinformatics
MScinBioinformatics Module2:CoreBioinformatics
Course2017-18 2StructuralBioinformatics.Lecture1.
• Smallmolecules• Lipids• Vitamins• Hormones• Neurotransmitters• Metabolites• …• combinationsandintersections
• Monomers,oligomersandpolymers• Aminoacids,oligopeptides (<20),polypeptides(<50),proteins• Monosaccharides,oligosaccharides(<10),polysaccharides• Nucleotides,oligonucleotides(<30,s-stranded),polynucleotides(s-stranded),
nucleicacids(DNA,RNA)• Isoprene,terpenes,polyterpenes (e.g.rubber)
Biomolecules
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StructuralBiology
Concernedwiththemolecularstructureofbiologicalmacromolecules,especiallyproteinsandnucleicacids,howtheyacquirethestructurestheyhave,andhowalterationsintheirstructuresaffecttheirfunction.
Hammerheadribozyme B-DNAMyoglobin
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Proteins:themostversatilemacromolecules
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Proteins:themostversatilemacromolecules
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Thereare20aminoacidscommoninproteins
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Aminoacids
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Aminoacids
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Proteinsarelinearpolymersofaminoacidsconnectedbyamidebonds
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Peptidebond
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Thepropertiesofthepeptidebondhaveimportanteffectsonthestabilityandflexibilityofpolypeptidechains
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Degreesoffreedomofthepolypeptidebackbone
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Therearefourlevelsofproteinstructure
Thenumberofproteinfolds(tertiarystructures)islargebutlimited
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Proteinstructurelevels
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Stericconstraintsdictatethepossibletypesofsecondarystructure
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Secondarystructureelements
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Secondarystructureelements
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Secondarystructureelements
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• Biologicallyrelevantmetalshaveahighenvironmentalavailability
• Manyalkali,alkaliearthandtransitionmetalsarenaturalbiometals:– Na,K,Mg,Ca– Mn,Fe,Co,Ni,Cu,Zn,...
• Theirinvolvementintheproteincanbe:– Folding– Functional
Metalions
Course2017-18 StructuralBioinformatics.Lecture1.
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• Inmanycases,livingorganismshaveundertakentheassimilationof organicorinorganicentitiestoincludespecificfunctionalityinproteins
Prostheticgroups
Course2017-18 StructuralBioinformatics.Lecture1.
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Foldedproteinsarestabilizedmainlybyweaknoncovalentinteractions
Formostproteinsthedifferenceinfreeenergybetweenthefoldedandunfoldedstatesissmall,i.e.onlyabout10timestheaveragethermalenergyavailableatphysiologicalconditions
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Proteinfoldingandstability
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Thefoldedstructureofaproteinisdirectlydeterminedbyitsprimarystructure
Thebalanceofself-interactions,interactionswithwater(andothercomponentsifpresent)andentropydrivesproteinfoldingandstabilizesthefoldedstructure
Itisallaboutthermodynamicsandkinetics,asusual
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Proteinfoldingandstability
Funnelmodelofproteinfolding
Dill&MacCallum.Science2012,338:1042
!ΔG = ΔH−TΔSHydrophobiccollapse
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X-raydiffraction
• AnX-raystrikinganelectronproducessecondarysphericalwavesemanatingfromtheelectron(elasticscattering)
• Aregulararrayofscatterers producesaregulararrayofsphericalwaves• Althoughthesewavescanceloneanotheroutinmostdirectionsthroughdestructive
interference,theyaddconstructivelyinafewspecificdirections,determinedbyBragg'slaw:
• Thesespecificdirectionsappearasspotsonthediffractionpatterncalledreflections• X-raysareusedtoproducethediffractionpatternbecausetheirwavelengthλ istypically
thesameorderofmagnitude(1–100Å)asthespacingd betweenplanes
!!2d sinθ = nλ
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Single-crystalX-raydiffraction
• AbeamofmonochromaticX-rayscollimatedtoasingledirectionstrikesasinglecrystal,producingscatteredbeams
• Thebeamsproduceadiffractionpatternofspots(reflections)onthedetector• Theintensitiesandanglesofthesebeamsarerecordedasthecrystalisgraduallyrotated• Multipledatasetsarecollected,witheachsetcoveringslightlymorethanhalfafull
rotationofthecrystalandtypicallycontainingtensofthousandsofreflections• Thesedataarecombinedcomputationallywithcomplementarychemicalinformationto
produceandrefineamodelofthearrangementofatomswithinthecrystal
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• Thedatacollectedfromadiffractionexperimentisareciprocalspacerepresentationofthecrystallattice
• Thepositionofeachdiffractionspotisgovernedbythesizeandshapeoftheunitcell,andtheinherentsymmetrywithinthecrystal
• Theintensityofeachdiffractionspotisproportionaltothesquareofthestructurefactoramplitude
• Thestructurefactorisacomplexnumbercontaininginformationrelatingtoboththeamplitudeandphaseofawave
• Inordertoobtainaninterpretableelectrondensitymap,bothamplitudeandphasemustbeknown
• Thephasecannotbedirectlyrecordedduringadiffractionexperiment• Initialphasescanbeassignedby:
• Ab initiophasingordirectmethods:smallmolecules• Molecularreplacement:knownrelatedstructure• AnomalousX-rayscattering:introducinganomalouslydiffractingatoms(e.g.SeMet)• Multipleisomorphous replacement:introducingheavyatoms
Thephaseproblem
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• Allisotopesthatcontainanoddnumberofprotonsand/orofneutronshaveanintrinsicmagneticmomentandangularmomentum(anon-zeronuclearspin),whichgivesrisetodifferentenergylevelsandresonancefrequenciesinamagneticfield
NMRinvolvestwosequentialsteps:• Thealignment(polarization)ofthemagneticnuclearspinsinanapplied,constant
magneticfieldH0
• Theperturbationofthisalignmentofthenuclearspinsbyemployinganelectro-magnetic,usuallyradiofrequencypulse.Therequiredperturbingfrequencyisdependentuponthestaticmagneticfield(H0)andthenucleiofobservation
• Electronicshieldingreducesthemagneticfieldatthenucleus(whichiswhatdeterminestheNMRfrequency).Asaresult,thefrequencyrequiredtoachieveresonanceisreduced:thisshiftintheNMRfrequencyiscalledchemicalshift
Nuclearmagneticresonance
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• Thesignalsareaconsequenceofrelaxationofthenucleitoequilibrium• Two-dimensionalNMRspectra(e.g.COSY,NOESY)provideinformationonthe
interactionsbetween1Hnucleithroughcovalentbonds(through-bondJcouplings)orthroughspace(nuclearOverhauser effect,NOE)
• Onlyatomswithin5Å ofeachothershowaNOE,anditsmagnitudevarieswiththedistancebetweenthem
• Thestructureoftheproteinmaybereconstructedfromasetofinter-proton(upper-bound)distancesinferredfromtheNOEintensities
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NMRspectroscopy
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Theresolutionofastructuregivesanideaofitsprecision.Theresolutionisdeterminedbytheamountofdatameasured:themoredatathehighertheresolution
• X-ray:adistanceinÅ indicatingthatanytwoatomsseparatedbymorethanaboutthisdistancewillappearasseparatemaximaintheelectrondensitycontourplot.1Å isatomicresolution
• NMR:doesnothaveaproperresolutionmeasure.Theatomic-positionroot-mean-squaredifferencebetweenthevariousmodelsgeneratedisgivenasanindicator
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Precisionofanexperimentalstructure
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X-raycrystallography:• Theproteinmustbecrystallisable• Itdoesnotprovideinformationondynamics
NMRspectroscopy:• Hasaprotein-sizelimit:normally70kDa,150kDa withTROSY• Theproteinmustbesolublewithoutaggregatingatconcentrationsclosetothoseofaproteininacrystallattice
• LessprecisethanX-raycrystallography(lessdataandlessprecisedata)
Both:• Theexperimentalconditionsarefarfromphysiologicalconditions:thestructuremaybeaffected
• Theremightbedisorderedregionsforwhichthestructurecannotbeassessed
• Theexperimentaldatahastobemanipulatedbeforeitcanbetransformedintocoordinates.Thismanipulationincludesanumberofassumptions(especiallyinNMR):experimentalstructuresarealsomodels
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Limitationsofexperimentalstructuredetermination
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• PolarresiduescanseetheirpKa substantiallyalteredwhenpassingfromawatermediumtoainnerproteinmedium
• Sincemanysimulationmodelshaveanexplicitatomicrepresentation,oneneedstohaveaprettygoodideaoftherealprotonationstate
• Wrongprotonationstatescanleadto:– Modifieddynamics– Wronghydrogen-bondingnetworkindockings– etc…
• Histidine isthemostimportantresiduetotakecareof• Asimpleneighbourhooddistanceanalysisisoftensufficientforaninitialguess.
ReasonablyaccuratepKa calculationswillbeinothercasesneeded.
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Theprotonationproblem
!!pKa = −log10Ka
!ka =
A−⎡⎣ ⎤⎦ H+⎡⎣ ⎤⎦HA⎡⎣ ⎤⎦
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pKas ofaminoacidsinwater
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Thefiguresinthissectionbelongto"ProteinStructureandFunction",byPetsko andRinge,NewSciencePress,2004,andweredownloadedfrom:http://www.new-science-press.com/browse/protein/resources/#illustrations
Otherrecommendedbooks:
"IntroductiontoProteinStructure",byBranden andTooze,GarlandPublishing,2ndedition,1999
"Proteins:StructuresandMolecularProperties",byCreighton,WHFreeman&Co.,2ndedition,1993
"StructureandMechanisminProteinScience:AGuidetoEnzymeCatalysisandProteinFolding",byFersht,WHFreeman&Co.,1999
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Bibliography
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3DproteinstructuresPDB,http://www.rcsb.org/pdb/CheckingstructuresPROCHECK,onlineathttp://www.ebi.ac.uk/pdbsum/ProteindatabaseUniProt,http://www.uniprot.org/StructuralclassificationofproteinsSCOP,http://scop.mrc-lmb.cam.ac.uk/scop/Proteinstructurecomparisonhttp://ekhidna2.biocenter.helsinki.fi/dali/DomainfamiliesPfam,http://pfam.xfam.org/SecondarystructurePSIPred,http://bioinf.cs.ucl.ac.uk/psipred/SmallcompoundsDrugBank,http://www.drugbank.ca/
Sitesfortheexercises