inless ofD III0010020AFIGUREIDiffractiondatafromorientedgapjunctionmembranesThepatternobtainedfrommouseliverspecimenL68isshownintheupperleftcornerafterquadrantaveragingsubtractionoffilmfogandc ID: 941263
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DiffractiondiagnosisofproteinfoldingingapjunctionconnexonsThomasT.Tibbitts,D.L.D.Caspar,W.C.Phillips,andD.A.Goodenough*RosenstielBasicMedicalSciencesResearchCenter,BrandeisUniversity,Waltham,Massachusetts02254-9110;and*DepartmentofAnatomyandCellularBiology,HarvardMedicalSchool,Boston,Massachusetts02115USAABSTRACTTodiagnosetheregularpolypeptideconformationingapjunc-tionmembranes,thex-rayintensitiesdiffractedfromorientedspecimenshavebeenseparatedintoamodulatedcomponentduetothecoherentlyorderedportionofthechannel-formingpairsofconnexon inless ofD. III00.100.20A-'FIGUREIDiffractiondatafromorientedgapjunctionmembranes.ThepatternobtainedfrommouseliverspecimenL68isshownintheupperleftcornerafterquadrantaveraging,subtractionoffilmfog,andconversiontocylindricalpolarcoordinates(R,Z)inreciprocalspace.Intensitynearthemeridianismissingduetothecurvatureofthesphereofreflection.Thesedatawereresolvedintotwocomponents:mo
dulatedintensityarisingfromcoherentlyorderedproteinwithintheconnexon(lowerright)anddiffusescatterfromdisorderedprotein,lipid,andwater(lowerleft).ThemodulatedcomponentwasangularlysmoothedbyconvolutionwithaGaussianof50standarddeviationtofurtherreducenoise(upperright).dlestructureshavediffractionprofileswhichcorrespondmostcloselywiththegapjunctiondata(Fig.2a).CylindricallyaveragedpatternsCylindricallyaverageddiffractionpatternsofimperfectlyaligneda-andfl-proteinswerecalculatedfromtheirFouriertransformstoassesshowtheorientationofstruc-turaldomainscouldbeinferredfromthegapjunctiondiffractiondata.Thebackbonesoftwo,B-andtwoa-proteinsareshownattheleftofFig.4,orientedtogiveamerid,ionaldiffractionmaximaat-4.8-4.9Aspacing;andtheircalculateddiffractionpatternsareshownattherightinFig.4,afterconvolutionwitha±150disorienta-tiondistributionandcylindricalaveraging.Thesharpnessofthemeridionalmaximaisrelatedtotheaxialex
tentoftheperiodicsecondarystructure.Toproduceamaximumonthemeridianbeyond0.2A'forferricytochromec'andTMVproteins,theorientationanglesofsomeofthea-helicesineachmoleculeweresetatangles�200tothereferencez-axis,withastandarddeviationof-+130fortherangeoftiltsoftheindividualsegments.Ifallfoura-heliceswerealignednearlyparalleltotheaxis,thenthemaximumofthe-5.4Apitchspacinglayerlinewouldhaveoccurredoffthemeridian.Thedistinguishingfeatureofthecylindricallyaver-ageda-helicaldiffraction,incontrasttothefl-sheetpatterns,isthedominantequatorialmaximumat11-12Aspacing.Theangularwidthofthismaximumisdeter-minedbytheorientationsofthea-helicalsegmentsrelativetothereferenceaxis,theorientationdistribution,andtheintrinsicoff-equatorialwidthofthediffraction(whichisrelatedtotheaxiallengthofthesegments).The-250angularhalf-widthofthe11-12Aspacingarcinthecomputeddisorientedpatternsiscomparablewiththatmeasuredinthegapjunction
patterns.Thus,abundleof1028BiophysicalJournalVolume57iin1028BiophysicalJournalVolume57May1990 SphericallyAveragedDiffractioncoc4._4-e)FIGURE2SphericallyaveragedintensitydistributionscalculatedfromFouriertransformsofproteinsrepresentingdifferentstructuralclasses.Gapjunctionsphericallyaverageddiffraction,shownineachframe,wasgeneratedfromthecylindricallysymmetricmodulatedintensity(Fig.I)byweightingeachgridpointbyitsdistanceRfromthemeridian,thenintegratingoverallanglestothemeridian.Theresultingcurvewaslow-passFourierfiltered(witha17-Aresolutioncutoff)andscaledwiththecalculatedcurvestohavethesameintegratedintensityinthe4-5Aspacingpeak.Thegapjunctionmaximanear10Afallsclosetocurvescalculatedfortwofour-helixbundleproteins,Rhodospirilliumferri-cytochromec'andtobaccomosaicvirus(TMV)coatprotein(A).Flavodoxin,aparallel,3-sheetstructuresurroundedbya-helices,andcarboxypeptidase,amixed-sheetstructuresurroundedbya-hel
ices,givemaximanear10Asmallerthanthatinthegapjunctiondata(B).Antiparallel,-sheetstructureshaveevensmallermaximanear10A,asshownbycurvescalculatedfortomatobushystuntvirus(TBSV)domain3,aneight-stranded13-barrel,andtheBenceJonesprotein,animmunoglobinconstantdomain13-barrel(C).a-helicessetatameanorientationangleA1of.200tothemembranenormal,withavariationof1O-150inthetiltanglesandadisorientationdistributionof±150,isconsis-tentwiththeangularwidthoftheequatorialarcsinthediffractionpatternrecordedfromthecoherentlyorderedproteininthegapjunctionconnexons.2.0-.9.coc1.0-Ca-hecalcontent(%)100FIGURE3Relationofthepeakintensityratioinsphericallyaverageddiffractionpatternstothea-helicalcontentoftheprotein.Theratioofaverageintensityintheinterval0.07-0.11A-'tothatintheinterval0.18-0.23A-iscorrelatedwithhelixcontentoftheorderedportionofthestructure(thoseresidueshavinganaverageDebyefactorofA2).(a)Immunoglobinlightchainconst
antdomain,asimpleantiparal-lel13-barrel(XuandSchiffer,1988)(b).Plastocyanin,alsoasimpleantiparallel,B-barrel(Gussetal.,1986).(c)Domain3ofTBSVcoatprotein,anantiparalleljellyroll13-barrel(Hopperetal.,1984).(d)Subtilisininhibitor,anantiparallel13-sheetwithtransversea-helix(Mitsuietal.,1979).(e)Carboxypeptidase,ana#mixed-sheetstruc-ture(Reesetal.,1983).(f)Flavodoxin,aparallel,8-sheetsurroundedbya-helices(Smithetal.,1977).(g)Lactatedehydrogenase,structur-allysimilartoflavodoxin(MusickandRossman,1979).(h)Insulinsubunit,asmalla13proteininthecubiccrystalform(Badgeretal.,manuscriptinpreparation).(i)Triosephosphateisomerase,aparallel,8-barrelsurroundedbya-helices(Banneretal.,1976).(j)TMVcoatprotein,anantiparallelup-downhelixbundle(NambaandStubbs,1986).(k)Ferricytochromec',anup-downdivergentbundleoffourantiparallela-helices(Finzeletal.,1985).(1)Hemoglobin,8-subunit,anantiparallelhelixbundle(BoltonandPerutz,1970).
(m)E.colirepressorofprimer(ROP),anantiparallelup-downfour-helixbundlewithtwofoldsymmetry(Banneretal.,1987).(n)Threestrandsofresidues96-115frominfluenzavirushemagglutinin,relatedbyathreefoldaxisinaparallel-strandedcoiledcoil(Wilsonetal.,1981).DiffractionfromtheconnexonpairanddodecamermodelsTosimulatethehighspatialfrequencymodulationofthegapjunctiondata,wehavecalculatedcylindricallyaver-aged,disorienteddiffractionpatternsofdodecamerswith622pointgroupsymmetry,builtfromthree-andfour-strandeda-helixproteinsarrangedtogivedimensionssimilartothechannel-formingpairofconnexonhexamers(Fig.5).Themodelswerebuiltwiththecentersofthea-helicaldomains42-44Aabovethemidplane,corre-spondingtothe42Adistanceofthegapjunctionbilayercenterfromthemiddleofthegap(Makowskietal.,1977,1984b);andthemeanradiiofthea-helixbundlesweresetTibbittsetal.DiffractionDiagnosisofProteinFolding1029GapJunctions.0DITibbittsetal.DiffractionDiagnosiso
fProteinFolding1029 area ofthe AA5.3423.05.8025.9havebeen EquatorialdataTheequatorialintensitydistributionisrelatedbythecylindricalBessellfunctionterms[J,(2irRri)ofordern=0,6,12...]totheradialcoordinatesr,,measuredfromthehexameraxis,ofthehelixsegments.Becausetheside-to-sidespacingofthea-helixsegmentsis-10-13A,themaximumintensitywilloccurnearR-0.09A-',withaspatialfrequencymodulationcharacteristicoftheradialcoordinates.Thewidthsofequatorialmaximainthediffractionpatternsoftheferricytochromec'andhemagglutinincoiled-coilmodels(Fig.5)aresimilarbecausethemeanradiiofthea-helicalsegmentsarer,24Ainbothmodels,eventhoughonemodelhasfoursegmentsandtheotherthree.Thesetwopatternsdifferinthepositionofthemaximumbecausethedistancebetweena-helixsegmentsislargerintheinfluenzacoiled-coilthaninferricytochromec'.ThepositionofthemaximumintheTMV-basedmodelisnear0.09A`andthearcwidthissimilartothatoftheothertwomodelpatterns(Fig.5
),althoughtherearedifferencesinthedetailsoftheintensitymodulationduetodetaileddiffer-encesinthestructures.ThegapjunctionpatternwasFourierfilteredtoremovethesharplatticesamplingfromthedataforcomparisonwiththemodeldiffractionpatterns(Fig.5).Thecorrespondenceintheoverallwidthofthe0.09A-'maximuminthegapjunctionpatternwiththatofthemodelequatorialdiffractionmaximaindicatesthattherearethreeorfournearaxiala-helixsegmentsintheconnexonsubunitwhicharelocatedatameanradius-r24Ainthehexamer.Tocomparetheprofileofthe0.09A'maximumfromthegapjunctionsandtheferricy-tochromec'-basedmodel,thepatternsweresmoothedwiththesamelow-passFourierfilter,sphericallyaver-agedandscaledintheinterval0.18-0.23A'(Fig.6a).Thesimilarityinwidthofthemaximumcharacteristicofthe24Ameanradiusisevidentfromthiscomparison.However,theslightlylowerintegratedintensityofthe0.09A'peakforthegapjunctionpatterncomparedtothatforthemodelferricytochromec'pattern
indicatesthatthea-helixcontentsareinproportiontothisintensityratio(cf.Fig.3).Theequatorialarcsinthemodeldisorientedpatterns(Fig.5)haveangularhalf-widthsofabout(&2+p2)1/2,where_isthehalf-widthofthedisorientationdistributionand41isthemeanorientationangleofthea-helicesrelativetothehexameraxis(Table1).Inthethree-strandedcoil-coilmodelpattern(Fig.5a),theangularhalf-widthoftheequatorialarcis,190correspondingtothe100averagehelixtiltconvolutedwiththe±150disorientationdistribution.Similarly,thearcwidthsfortheTMVproteinandferricytochromec'patternsare230.(1ca:SphericalyAveragedDifraction11REquatorialArcsGapJunctionsICD\Ferricytochromec'.IDodecamerd:coOrientX=nX=ODegrees90FIGURE6Sphericallyaverageddiffraction(A)andtheangulardistri-butionofequatorialintensity(B)inthecylindricallyaveragedpatternfromapairofferricytochromec'hexamerscomparedtocurvesfromthegapjunctiondata.AlOOAresolutionlow-passFourierfilterwasappliedt
othesphericallyaveragedferricytochromec'dodecamerintensitydistributionandtothesphericallyaverageddiffractiondata;thisremovedtheeffectsofhexagonallatticesamplinginthedata.Theangularspreadofthegapjunctiondisorientationdistribution(showninBasmeasuredfromthesmall-anglemeridionalfringes)dependsonlyupontheorientationofthemembranes,andisnotasbroadasIIAspacingequatorialarcsinthedata.Theangularspreadoftheequatorialarcsinthemodel,whichissimilartothatofthegapjunctiondata,isduetothe230meantiltofthea-helicescombinedwiththe150standarddeviationoftheorientationdistribution.and270correspondingrespectivelytomeana-helixtiltsof18°and230.Theangulardistributionoftheequatorialmaximuminthegapjunctionpatterniscomparedwiththatoftheferricytochromec'modelinFig.6b.Becausethehalf-widthsofthesetwopatternsaresimilar,itappearsthatthea-helicesintheconnexon,likethoseinthemodel,areorientedatameanangleof=200tothehexameraxis.Thespreadofor
ientationsintheconnexonappearstobenarrowerthanintheferricytochromec'model,sincethemeasuredintensitydistributionfortheequatorialarcfromthegapjunctionsliesabovethatforthesimulatedpatternsatanglessmallerthanthehalf-widthandbelowatlargerangles;fromthiscomparisonoftheshapesofthecurves,thestandarddeviationforthetiltanglesintheconnexonisestimatedtobe-10°.Summaryofa-helicalparametersAbout60%oftheorderedportionoftheconnexonisa-helical,asindicatedbytheratioofintensityinthe0.09and0.20A-'spacingpeaksinthesphericallyaverageddiffractiondata.Inthecylindricallyaveragedpattern,theoverallspreadofthemeridionalfringesintheaxialdirectionbetweenZ=0.18and0.24A'indicatesthatthemeanlengthofLofthehelixsegmentsis-35Aandthemaximumhelixsegmentlengthis=40A.Fromtheaveragewidthofthemeridionalfringes,themeandis-Tibbittsetal.DiffractionDiagnosisofProteinFolding1033Tibbittsetal.DiffractionDiagnosisofProteinFolding1033 ofthe channelbe D