MSc in Bioinformatics - UABbioinf.uab.es/xavier/Module2-StructBiol-Lecture1.pdfMSc in Bioinformatics...

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



StructuralBiology

Concernedwiththemolecularstructureofbiologicalmacromolecules,especiallyproteinsandnucleicacids,howtheyacquirethestructurestheyhave,andhowalterationsintheirstructuresaffecttheirfunction.

Hammerheadribozyme B-DNAMyoglobin



Proteins:themostversatilemacromolecules


Thereare20aminoacidscommoninproteins


Aminoacids



Aminoacids


Proteinsarelinearpolymersofaminoacidsconnectedbyamidebonds


Peptidebond


Thepropertiesofthepeptidebondhaveimportanteffectsonthestabilityandflexibilityofpolypeptidechains


Degreesoffreedomofthepolypeptidebackbone


Therearefourlevelsofproteinstructure

Thenumberofproteinfolds(tertiarystructures)islargebutlimited


Proteinstructurelevels


Stericconstraintsdictatethepossibletypesofsecondarystructure


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.


Foldedproteinsarestabilizedmainlybyweaknoncovalentinteractions

Formostproteinsthedifferenceinfreeenergybetweenthefoldedandunfoldedstatesissmall,i.e.onlyabout10timestheaveragethermalenergyavailableatphysiologicalconditions


Proteinfoldingandstability


Thefoldedstructureofaproteinisdirectlydeterminedbyitsprimarystructure

Thebalanceofself-interactions,interactionswithwater(andothercomponentsifpresent)andentropydrivesproteinfoldingandstabilizesthefoldedstructure

Itisallaboutthermodynamicsandkinetics,asusual


Proteinfoldingandstability

Funnelmodelofproteinfolding

Dill&MacCallum.Science2012,338:1042

!ΔG = ΔH−TΔSHydrophobiccollapse



X-raydiffraction

• AnX-raystrikinganelectronproducessecondarysphericalwavesemanatingfromtheelectron(elasticscattering)

• Aregulararrayofscatterers producesaregulararrayofsphericalwaves• Althoughthesewavescanceloneanotheroutinmostdirectionsthroughdestructive

interference,theyaddconstructivelyinafewspecificdirections,determinedbyBragg'slaw:

• Thesespecificdirectionsappearasspotsonthediffractionpatterncalledreflections• X-raysareusedtoproducethediffractionpatternbecausetheirwavelengthλ istypically

thesameorderofmagnitude(1–100Å)asthespacingd betweenplanes

!!2d sinθ = nλ



Single-crystalX-raydiffraction

• AbeamofmonochromaticX-rayscollimatedtoasingledirectionstrikesasinglecrystal,producingscatteredbeams

• Thebeamsproduceadiffractionpatternofspots(reflections)onthedetector• Theintensitiesandanglesofthesebeamsarerecordedasthecrystalisgraduallyrotated• Multipledatasetsarecollected,witheachsetcoveringslightlymorethanhalfafull

rotationofthecrystalandtypicallycontainingtensofthousandsofreflections• Thesedataarecombinedcomputationallywithcomplementarychemicalinformationto

produceandrefineamodelofthearrangementofatomswithinthecrystal



• Thedatacollectedfromadiffractionexperimentisareciprocalspacerepresentationofthecrystallattice

• Thepositionofeachdiffractionspotisgovernedbythesizeandshapeoftheunitcell,andtheinherentsymmetrywithinthecrystal

• Theintensityofeachdiffractionspotisproportionaltothesquareofthestructurefactoramplitude

• Thestructurefactorisacomplexnumbercontaininginformationrelatingtoboththeamplitudeandphaseofawave

• Inordertoobtainaninterpretableelectrondensitymap,bothamplitudeandphasemustbeknown

• Thephasecannotbedirectlyrecordedduringadiffractionexperiment• Initialphasescanbeassignedby:

• Ab initiophasingordirectmethods:smallmolecules• Molecularreplacement:knownrelatedstructure• AnomalousX-rayscattering:introducinganomalouslydiffractingatoms(e.g.SeMet)• Multipleisomorphous replacement:introducingheavyatoms

Thephaseproblem



• Allisotopesthatcontainanoddnumberofprotonsand/orofneutronshaveanintrinsicmagneticmomentandangularmomentum(anon-zeronuclearspin),whichgivesrisetodifferentenergylevelsandresonancefrequenciesinamagneticfield

NMRinvolvestwosequentialsteps:• Thealignment(polarization)ofthemagneticnuclearspinsinanapplied,constant

magneticfieldH0

• Theperturbationofthisalignmentofthenuclearspinsbyemployinganelectro-magnetic,usuallyradiofrequencypulse.Therequiredperturbingfrequencyisdependentuponthestaticmagneticfield(H0)andthenucleiofobservation

• Electronicshieldingreducesthemagneticfieldatthenucleus(whichiswhatdeterminestheNMRfrequency).Asaresult,thefrequencyrequiredtoachieveresonanceisreduced:thisshiftintheNMRfrequencyiscalledchemicalshift

Nuclearmagneticresonance


• Thesignalsareaconsequenceofrelaxationofthenucleitoequilibrium• Two-dimensionalNMRspectra(e.g.COSY,NOESY)provideinformationonthe

interactionsbetween1Hnucleithroughcovalentbonds(through-bondJcouplings)orthroughspace(nuclearOverhauser effect,NOE)

• Onlyatomswithin5Å ofeachothershowaNOE,anditsmagnitudevarieswiththedistancebetweenthem

• Thestructureoftheproteinmaybereconstructedfromasetofinter-proton(upper-bound)distancesinferredfromtheNOEintensities


NMRspectroscopy


Theresolutionofastructuregivesanideaofitsprecision.Theresolutionisdeterminedbytheamountofdatameasured:themoredatathehighertheresolution

• X-ray:adistanceinÅ indicatingthatanytwoatomsseparatedbymorethanaboutthisdistancewillappearasseparatemaximaintheelectrondensitycontourplot.1Å isatomicresolution

• NMR:doesnothaveaproperresolutionmeasure.Theatomic-positionroot-mean-squaredifferencebetweenthevariousmodelsgeneratedisgivenasanindicator


Precisionofanexperimentalstructure


X-raycrystallography:• Theproteinmustbecrystallisable• Itdoesnotprovideinformationondynamics

NMRspectroscopy:• Hasaprotein-sizelimit:normally70kDa,150kDa withTROSY• Theproteinmustbesolublewithoutaggregatingatconcentrationsclosetothoseofaproteininacrystallattice

• LessprecisethanX-raycrystallography(lessdataandlessprecisedata)

Both:• Theexperimentalconditionsarefarfromphysiologicalconditions:thestructuremaybeaffected

• Theremightbedisorderedregionsforwhichthestructurecannotbeassessed

• Theexperimentaldatahastobemanipulatedbeforeitcanbetransformedintocoordinates.Thismanipulationincludesanumberofassumptions(especiallyinNMR):experimentalstructuresarealsomodels


Limitationsofexperimentalstructuredetermination


• PolarresiduescanseetheirpKa substantiallyalteredwhenpassingfromawatermediumtoainnerproteinmedium

• Sincemanysimulationmodelshaveanexplicitatomicrepresentation,oneneedstohaveaprettygoodideaoftherealprotonationstate

• Wrongprotonationstatescanleadto:– Modifieddynamics– Wronghydrogen-bondingnetworkindockings– etc…

• Histidine isthemostimportantresiduetotakecareof• Asimpleneighbourhooddistanceanalysisisoftensufficientforaninitialguess.

ReasonablyaccuratepKa calculationswillbeinothercasesneeded.


Theprotonationproblem

!!pKa = −log10Ka

!ka =

A−⎡⎣ ⎤⎦ H+⎡⎣ ⎤⎦HA⎡⎣ ⎤⎦



pKas ofaminoacidsinwater


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


Bibliography



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/

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