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ArrestinexperimentsTheterminationstepinGPCRsignalinginvolvesatwo-stepmechanism-arapidphosphorylationofthereceptorfollowedbythearrestinbindingthatstericallyblocksfurtherinteractionofG-proteinwiththereceptor,resultinginterminationoftheprimarysignalingevent.WehavepreviouslyreportedcrystalstructuresandSAXSstudiesonconstitutivelyactivevariantsofvisualarrestin(p44-anaturallyoccurringtruncatedvariant;andR175E-achargereversalarr-1mutantinthepolarcore).Currently, we are investigating the effect of synthetic peptides that mimic thephosphorylated C-terminus of rhodopsin on arr-1 variants. Our preliminary resultsusing size-exclusion chromatography (SEC) show complex formation and a shift inelutionprofile,suggestingachangeinoligomericstate.IntheSAXSexperimentonBM29wehaveinvestigatedstructuralchangesuponbindingofasyntheticpeptidethatactivatesarrestines.Belowisasummaryofthekeyresults:Ascontrolswehavemeasuredtheproteinsp44andR175Ewithoutthepeptidebeingpresent(fig1).Thecontrolexperimentsdemonstratedthatbothproteinsarepresentinsolution.P44showsabsenceofaggregation,R175Ehadasmallcorrectableamountofaggregation.
Figure1:SAXSdataofp44andR175Eintheabsenceofsyntheticpeptide.Theredsolidlinearecalculatedtheoreticalscatteringcurvesbasedontheavailablecrystalstructuresofp44andR175E,respectively.
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Inasecondstepweaddedthesyntheticpeptidetothep44andR175EsolutionsandrecordedSAXSdata.Theexperimentaldataisshowninfig.2.
Figure2:MeasuredSAXSdataofp44andR175inthepresenceofpeptide.Thesolidlinesaremodelsofdimericproteins.
Themeasuredforwardscatteringdemonstratesdirectlythatpeptidebindinginducestheassemblyofp44andR175Edimers.Possibleassembliesofdimershavebeenscreenedandwecoulddeterminetopossibledimerisationassemblies,whichareactuallyverysimilarintheiroverallshape,butdifferintherotationofthemonomersaroundthelongaxis.
KineticexperimentsofLOVproteinsafterlight-excitationOur group members have determined crystal structures of, PpSB1-LOV, DsLOV ofDinoroseobacter shibae DFL12T, and more recently, dark state crystal structure ofPpSB1-LOV. Comparison of the two states reveal remarkably large conformationalchanges including~11Åmovementof theC-terminalhelix Jα,disruptionofhydrogenbondsinthedimerinterface,anda~29°rotationofchainBrelativetochainA.Here,wewouldliketoperformtime-resolvedkineticmeasurementsofdarkrecoveryofPpSB1-LOVproteinvariantsusingSAXS.WehaveperformedkineticSAXSexperimentsofPbSB1-R66I.TheproteinsolutionwasilluminatedbybluelightandtherecoveringprocessinthedarkwasfollowedbykineticSAXSexperiments.ThedifferenceSAXSspectrum(lightilluminatedatt=0minusdarkatt)atdifferenttimesisshowninfigure3.Acharacteristicdropisfoundat0.05Å-1,whichwasalsoobservedinstaticSAXSexperimentperformedbeforeonBM29.
Figure3:DifferenceSAXSspectrumoflight-illuminatedR66Iminusproteininthedarkatdifferenttime-points.
Thedifferencedatawereintegratedovertheq-rangeandtheintegralisplottedasafunctionoftimeinfigure4.Thesolidlineinisaexponentialwiththerelaxationtimeof24minthathasbeendeterminedwithUV/visabsorption.Thekineticexperimentsclearlyshowthatstructuralchangesinresponsetodarkrecoveryareobserved.Thetimedependenceseemstofollowthepreviouslydeterminedrecoverytime.Thedata,however,arenoisyanddonotallowanunbiasedfittodeterminetherelaxationtimewithunbiasedaccuracy.
Figure4:ChangesinthedifferenceSAXSsignal(light–dark)asafunctionoftime.Thesolidlineisanexponentialwithrelaxationtimeof24min.
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