THE ROLEOF KEY AMINO ACIDS IN THE COLD-ADAPTATION OF THE COILED-COIL
PROTEIN TROPOMYOSIN
ByKorrinaRFudge
A thesissubmitted in partialfulfillmentof the requirements for the degree of Master's of Science
Department of Biochemistry Memorial University of Newfoundland
December,20ll
St.Joh n's Newfoundland
Abstract
Thestructural basis of cold-adaptation ina rod-shaped(a-helica l,coiled-co il)protein tropom yosinwasinvestigatedby site-directed mutagenesis.Ana-type trop omyosin from Atlanticsalmo nskeletal musclehavingtwent y aminoacidsubstitutionscompared to a warm-blooded counterp art (rabbit) wasused as amodel. Two adaptive-strategieswere elucidated.Theconformationalstabilityoftropom yosin wasshown to beenhanced bythe presence of apolar aminoacid, threonine-I 79, within thehydrophob ic coreand the presence of apair ofclosely-spaced glycinesat positions 24and27. The spec ific details are outlined belowin pointform :
I)Fourmutant s ofAtlanticsalmonfastskeletal muscle alpha- tropomyosi nwere engineered usingtheQuikChangeLightnin g site directedmutagenesiskit.Mutations werechose n inorder to investigatetheroleofapair ofunique glyc ines neartothe amino- terminal endas wellasthreonin e179which occurs ina core positionofthe coiledcoil.
Thefour mutant swere:Gly24Ala,Gly27Ala, Thrl79Alaandadoubl emutant, Gly24A lalG ly27A la. Mutations were confirm edby DNA sequencing.
2)Recomb inant (mutatedand non-mut ated)tropom yosin s wereobtained by expressio n in BL2 1 cells and inductionwith isoprop ylthiogalactopyr anoside. Enrichedproteinwas isolated,withoutexposure toorganic solventsor hightemp eratures,by salt-indu ced precipitation and chrom atography onionexchangecolumnand hydro xyapaptit e columns.
3) Far-UVcircular dichroism wasusedto investigatetheconfo rmat iona lstabili tyof the recombinanttrop omyosins (0.1M NaCI,20mM sodiumphosphate,1-2 mM dithiothreito l, 0.01%mass/volNaN) .The observed meltingtemperat uresof the three glycinemutants were similar to eachotherandthatof the non-mut atedrecombinanttropom yosin:
Gly24A la,36.9 °C; Gly27Ala, 37.3 °C; Gly24Ala Gly27A la,38.1°C andnon-mutated, 37.0°C. However,the Thrl79A la mutant showe dsignifica ntstabi lization,40.7°C.
Melting profiles ofthefourtropom yosinmutants and non-mutat edrecombinant tropomyo sin showed that thefourmutant sdispla yedmore cooperative unfoldin gprofiles compare d to the non -mut atedprotein.
4) Limite dchymotrypsin digestion (buffer:50 mM NH4HCO) ,0.1M NaCl,ImMOTT, pH8.5),asmoni tored by SOS PAGE,revealedthat the non- mutated tropomyosinismore susceptibleto proteolysisthan the Thrl79Ala mutant (37°C,-I:500 enzyme:s ubstrate mole ratio). After 30min, noneof theintactnon-mu tated protein was detecte din the reactionmixture whereasnotalloftheTh rl79Alamutant had been digested.
At -25 and-10°C,thedifferenceinthe rate ofdigestion betwee n the two, wasnot as significa nt.
5)From po ints3and 4 itcan bcconcluded that thrco nine in the 179position of salmo n tropom yosinisdestabili zin gcomp aredtothe alanine inthesame positi onofrabbit skeletalalpha-tropomyos in.
6) Sequencingoftwofragment s from the chymo tryps in digestion ofnon-mu tant recombin anttrop om yosinindicatesthatthe initialcleavagesiteisbetweenLeuIIand Lysl2.
7) Omp-T digestion (buffe r:0.1 MNaCI,50 mM sodiumphosphate,S mM EDTA,I mM DTT,pH 7.0)patterns, asmonit oredby SDS PAGE, were compared between Gly24A la, Gly27A la,Gly24A laGly27A la, and nonmutated tropomyosi nat-10,-25 or -37°C.At all threetemp eratures,non-mut atedtropomyosin wasdigested fastercompared to the glycine mutant s, whichwerediffere nt to eachother.Theobserve drate ofbreakdown decreased inthe order:Gly27A la>Gly24A la>Gly24A laGly27A la, indicating that both glycinesinflue nce the conformationalstabilityofthe amino -termi nalregionand that Gly24A laismoreinfluenti althan Gly27Ala.
Note: Someofthe abovefindings were reportedin prelimi naryform at this year's Biophysica lSociety Meetin g (Fudge, K.R.and Heeley, D.H(20 11)Amutant of Atlantic salmonfastmuscletrop om yosin.ss"Biophysica lSociety(Baltimore)).A full manuscript is in preparationfor submissio n toBiochemi stry.
Acknowledge ments
First,Iwould liketothankmy superviso r, Dr. DavidHeeley, forallowing me the opportunity to workis in hislab and forhis guidance,encourage mentandadvice throughoutthisthesis.Iwouldalso liketothankmy supervisorycommittee, Dr. Ross McGowan and Dr.MikeMorrowfortheir suggestionsandguidance throughoutthis researchproject.I wouldliketo express my gratitude forthe staffatCREA IT-Dr.
Elizabeth Perry and Dr.Ed Yaskowiak -fortheirhelpandadviceon DNA sequencing.I amgratefulto Ms.DonnaJackman for herassistance andsuggestionsin theinitialstages of myresearch.Iwould like tothankDr. MikeHayleyforhis advice oncircular dichroi sm andhishelp increating the graphs. My appreciationgoestoMr.Craig Skinner forhistechnicalsupport throughoutthisresearchproject.Iamgrateful tothe technicians attheAdvanced Protein TechnologyCenter(SickChildren'sHospital,Toronto) for sequencing proteinfragm ents.Iwould liketo express my gratitude tomyformerlab mate,Robert Mercer,for his suggestionswhen Ifirststarted my research.Iwantto thank mylabmate,Tolu, forallof the help,supportandadviceshe has given me,bothinside andoutsideofthelab.Iwould liketo acknowledgeNSERCandthe School of Graduate Studiesat Memorial Univers ityof Newfoundland forproviding funding for this research endeavo r. Finally,I wouldliketothank myparents,brother, sisterand friends forallof theirsupport throughoutthepast coupleofyears-without you, I never would havemade it this far.
I
Tableof Content s
Abstract Acknowledgements Table of contents Listof tables Listof figures List of abbreviations
Ch a p ter I Introduction 1.1 Struc tureof striated muscle 1.2 Mecha nis mof contract ion 1.3 Majorsarcomericproteins
1.3. 1 Myosin 1.3.2 Actin 1.3.3 Troponin
1.3.3. 1TroponinC 1.3.3.2Troponinl 1.3.3.3TroponinT 1.3.4 Tropomyosin
1.3.4.1Tropomyosinisoforms 1.3.4.2Post-translationalmodifications 1.3.4 .3Interactions with selfand other proteins 1.4 Atlantic Salmon tropomyosin
1.5 Sites of proteolytic digestion in tropomyosin 1.6 Psychrophilic proteins
1.7 Mutanttropomyosins 1.8 Goalsof study Ch a p ter 2 Mat erialsand Mcthods
2.1 pHmeasur ements 2.2 Electro phores is 2.3 Dialysis 2.4 Spectroscopy
2.4.1 UV-Visib leabso rbance 2.4.2 Circular dichroi sm
iv
vii viii
2 5 7 7 9 II 12 12 13 14 18 19 20 22 23 25 26 27 29 29 29 30 30 30 31
2.4.3 Nanodrop 32
2.5 Site-directed mutagenesis 32
2.5.1 Oligonucleotideprimers 32
2.5.2 Purification ofpTrc99Aplasmidfrom BL21 cells 33
2.5.3 Site-direc ted mutagenesis 33
2.5.4 Transformation ofDNA withXLIO-Gold ultracompetent cells 34
2.5.5 TransformationofDNA withBL21 cells 35
2.6 DNA sequencing 35
2.7 Expressio noftropomyosinin bacteria 37
2.8 Enrichmentoftropomyosin 38
2.8.1 lon exchange chromatography 38
2.8.2 Hydroxyapatite chromatography 39
2.9 Proteolyticdigestion 39
2.9.1 Omp-Tdigestion 39
2.9.2 Chymotrypsin digestion 40
2.10 WesternBlotting 41
2.11 Calculating mean residue ellipticity 41
Chapter 3 Results and Discussion 42
3.1 Purification ofDNA from BL21plasmids 42
3.2 Nucleot idesequencing 42
3.3 Expressionoftropomyosin 45
3.4 Heat-inducedunfold ing 52
3.5 Chymo trypsi n digestion 60
3.6 Sequencingof chymo trypsinfragments 69
3.7 Omp-T digestion 71
Chapter 4 Conclusions and Future Directions 83
4.1 Conclusions 83
4.2 Future directions 89
References 92
List of Tables
Table I Differencesbetweenthe rabbitandsalmon fastmuscletropom yosin 24. Table2 Average melt ingtemp eratures andstandard deviations ofthenon- 58
mutant and mutant salmon tropomyosins
List of Figures
Figure I Thearchitectureofthe striated muscle
Figure2 Diagram of the sliding filamenttheory ofmuscle contraction
Figure3 Thestruc tureoftropomosyi n 15
Figure 4 Confirma tionof the Gly24A la trop om yosinmutation compared to 43 the cDNAof Atlanticsalmon fastmuscletropom yosin
Figure S Confirma tionoftheGly27Alatropom yosinmutationcomp aredto 44 the cDNA ofAtlanticsalmon fastmuscletropom yosin
Figure6 Confirma tion ofthe Gly24AlaGly 27Alatropom yosinmutation 46 compared to the cDNA of Atlantic salmonfast muscletropom yosin Figur e 7 Confirma tionof the Thrl79Atropomyosin mutation compared to 47
the cDNA ofAtlanticsalmon fastmuscletropom yosin
Figure8 SDS PAGEanalysis ofa lysateofIPTG-inducedBL21 cells 48 containingGly24A la tropomyosin
Figure9 QSepharoseFastFlow chromatography profil e of crude 49 recomb inanttropom yosin
Figure 10 Hydr oxyapatit e chro matograp hy profile ofrecombin ant 50 trop om yosin
FigureII SDS PAGE analysisofhighl y-enri chedmutant andnon-mu tant 51 tropom yosins
Figure12 Normalizedcurveofthemeltingprofiles ofthree samplesof 53 Gly24A la tro pomyosi n
Figure 13 Norma lize dcurveofthemeltin gprofiles of eightsamplesof 54 Gly27A la tropom yosin
Figur e14 Normalized curve of themeltingprofiles ofeightsamplesof 55 Gly24AlaGl y27Alatropomyosin
Figure 15 Norm alized curve of themeltingprofiles ofeightsamplesof 56 Thrl79Alatropomyosin
Figure16 Normali zed curveof the meltingprofiles ofeightsamplesofnon- 57 mutanttropom yosin
Figure17 Com pa riso noftheaveraged ,norm alized meltingcurvesof the three 61 glyci ne mutant s
Figure 18 Com pariso nof theaveraged,norm alizedmelt ing curvesoftwo 62 glyci ne mutants and thenon-mut ant tro pomyos in
Figure19 Compariso nofthe averaged,norm alizedmelt ing curvesof the 63 double glyc ine mutant and thenon-mut ant tropomyosi n
Figure20 Compa riso nof theaveraged,norm al izedmelting curvesof the 64 threon inemutant and thenon-mut ant tropomyosi n
Figure 21 Com pariso nofthe susceptibilityofnon-mutantand Thr l79A la 65 trop om yosinstolimited chymot ryp ticdigestionat37°C
Figure 22 Compariso nofthe suscep tibilityofnon-mut ant and Thr 179Al a 67 trop om yosin stolimited chymotrypticdigestionat 25°C
Figure 23 Comparisonofthe susc eptibilityof non-mutant andThr l79A la 68 trop om yosins tolimited chymotrypticdigestionat 10°C
Figure24 Comparison oftheOmp-Tdigestion ofnon-mu tant andGly24A la 72 trop om yosins at 10"C as analyse d by SDS PAGE
Figure25 Compa riso nofthe Omp -T digestion ofnon -mu tant andGly24A la 73 trop om yosins at25°Cas ana lyse d by SDS PAGE
Figure 26 Compa riso nof the Omp-T digestion ofnon-mu tant andGly24A la 74 trop om yosin at 37°C
Figure27 Compa riso nofthe Omp-Tdigestio nof non-m utan tandGly27A la 76 tropomyos inat10°C
Figure28 Compa riso nof theOmp-Tdigestion of non-m utantandGly27A la 77 tropom yosin at 25°C
Figure29 Compariso nofthe Omp-T digestion of non-mutantandGly27A la 78 tropom yosin at 37°C
Figure30 Com par iso nofthe suscep tibilityofthethree glycinemutant 79 trop om yosinsto Omp-Tdigestion at25°C
Figure31 Com par iso nof thesuscep tibili tyofthethree glyci ne mutant 81 trop om yosinsto Omp-T digestion at10°C
ADP ATP
~-ME CAPS eDNA dl-hO DNA OTT EDTA EGTA E28ol mg/ml IPTG LB MOPS mRNA MWCO NMR Omp-T PCR pI PMSF rpm RT SDS SDS PAGE SOC TEMED TnC TnI TnT Tris UV
List of Abbreviations Adenos ine diphosphate
Adenosinetriphosphate
~-mereaptoethanol
N-eyclo hexy l-3-aminopro panesulfonicae id Complime ntary deoxyr ibonucle icae id Deion ized water
Deoxyrib onucleic acid Dithiothr eitol
Ethylene dia mine tetraacetic acid Ethyleneglyeo l tetraaceti c acid
Extinctionc oeffieient ofa I mg/mlprotein at280 nm Isopropyl~-D-lthiogalactopyranosid e Luria broth
3-(N-morpholino)propanesulfonie acid Messengerribonucleicaeid Molecularweight cutoff Nuclearmagneti cresonance Outermembran eproteaseT Polym erase chainreaetion Isoeleetricprecipit ationpoint phen ylmeth ylsulfon ylfluoride Revolu tionsperminute Roomtemp erature Sodium dodecy ls ulfate
Sod iumdodeeyl sulfate polyacrylam ide gelelectro phores is SuperOptimalbroth withCatabolite repression N, N, N', N'-tetramethylethylenediaminutese TroponinC
Tropon inl Tropon inT
Tris (hydroxyme thyl)aminomethane Ultraviolet
Chapter 1 Introduction
Thepurposeof thisthesis was to establisha rolefor key amino acids in the cold- adaptation of therod-shaped protein, tropomyosin.Tropomyosin is an a-he licalcoiled- coil andan importa nt regul atory componen tin the musclethinfilament.This research used as a model trop om yosin fromAtlanticSalmon.Four recombinant mutants were preparedbyQuik changelightnin g site-direc ted mutagenesis.Mutations focused onone site(spec ifica lly, residu e-I79) within the almost-continuous hydroph ob ic seamthatis principall yresponsibl e for stab ilising the coiled-co ilas wellas apair of glycin es (residues 24 and27) in the amino termin alregion thataresolventexposed.Tempera tureinduced unfo ldingexperime ntsinvo lvingcircular dichroism showe d that changi ngoneamino acid (specifically,threonine-I 79to alanine)couldsignificantlyaffect theconformational stability oftropom yosin.The spectroscopic findings were confirmedby limited proteolytic digestion (chymotrypsinand outermembrane protease T) and extended by Edman-basedsequencingof fragments inordertolocate sites havingaltered molecu lar flexibility.
Theintrodu ction ofthe thesiswill start bybriefl ydescribin gthe struc tureofthe stria ted muscle and the mechanismofmuscle contraction.Thethesis willthendescribe themajorprotein sin the sarco mere: myosin , actin, troponin and trop om yosin . Follow ing thebriefintrodu ction of theproteins,tropomyo sinwill be expl ainedin moredetail incl uding its aminoacidsequenceand the sitesof chymo tryps inand Omp-T digestion.
Thethesis will then outlinesomeofthe current knowledge ofpsyc hro philic(low- temp eratur e)protein s,thereasoningbehind preparin gachose n tropomyosinmutant , and adepiction ofthe goalsofthe thes is.
1.1 Structureof Striated Muscle
Muscle is contrac tile tissuethatisresponsibl e for move men t inanimals. It is comp osedof spec ializedcells which are responsiblefor itscontractionand regulation.
Muscle tissueshave ada pted toperform a varietyoffunction s whic h has resultedin three types ofmuscletissue: smooth,cardiacandskeletal. Smoothmuscleisnon-striated and is responsible for involunt arycontraction in the wallsofblood vessels,respiratory tract and gastroi ntestinal tract. Cardiac muscle is striated musclethatis responsiblefor the involuntarycontrac tionsof theheart. Skeletal muscle is stria tedmuscle thatis responsible for voluntarycontract ion.
Muscle sarcomeres(Figure lA), whicharegenerally2-3 urn long and 1-2urn in diame ter, link end-to-e ndinorder to form long thinstrandscalled myofibrils (Squire, 1997,review).Thesemyofibril srun parallelto each other andformamusclefibre (Squire,1997,review).At highmagnifi cation,themyofibr ils appearstriated due tothe overlapping thick(15nm diameter) and thin filament s (6nm diam eter).Thethin filament sare comprisedof actin ,tropom yosin and troponin and thethickfilam ents are madeupofmyosin (Figure I Band IC).Located at eitherendofthe sarcomereis the Z line which links success ivesarcomereswithin themyofibr ils.Italso serves asthe attachment point ofthethinfilame nts.In betweentwo Zlines are twoIbandswhic hare
COPI"I!t'0200SBienI InC .... mllgS
I
HIAOTOTAILOVliLA"
E 0'TIIO"OMYOall.
Heeley"l~l. 1987Jour Bio IChem262:9971.9978
Figure1: The architectureof theskeletal muscle
A schematicrepresentationof (A) the muscle fibre, (8) the myofibril,(C) the sarcomere, (D) thethickfilament and(E)themaj orproteins of thethinfilaments.
oneither side of an A band.TheA band isdarkerthan theIbandbecausethe forrner is comprisedof overlappin gthick and thin filaments whereas thelatteris compri sed ofthi n filament sonly (Huxleyand Hanson,1954;Aidley ,1991,pg248-249 ,review)(FigureIB and IC).
Skeletalmusclehasbeendividedinto three catego ries basedon the followin g criteria:numberofmitochondriapresent,importan ceofoxid ation andglyco lytic pathwa ysin respir ation,myosin ATPaseactivity, contractiontime of thefibres and the fibresresistanc etofatigue.The threetypes ofske letal muscl eare slowoxidative, fast glyco lyticand fast oxidative fibres.Slow oxidative fibreshavea highmitochondri on content, lowglycogencontent ,and a long contraction time (Barnardetal.,1971).Their redcolour isdueto thehigh concentrations of myoglobin ,theyare sma ll in diameter and theycontain ahigher supply of oxygen sotheycansustainaerobicactivity.In compariso n,the fastglycolytic fibreshavea high glycoge ncontent,shortcontraction time andalowmitochondri on content. Theyhave alargerdiameter and have alighter colour dueto adecreasedamountofmyoglobin .Thefast oxidative fibres arean intermed iate of theother twofibres.Thesefibreshave ahigh mitochondr ion content, high glycoge n cont ent and ashort contra ctiontime.They also have ahighresistancetofatigue andare reddueto theirhighermyoglobin content. Thefastoxid ati ve andfast glyco lyticfibres constitute themajorit y ofskeletal musclein vertebr ates (Barna rdetal.,1971).
1.2Mechanism of Contraction
Both thetrop onincomplex (troponinC,tro ponin Iand trop onin T) and tropomyosin are involved in theregul ation ofmuscle contrac tion,although the exact mechanismisnot yet known.According tothe steric blockingmodel,tropom yosin completelycovers themyosinbinding siteonactinin therelaxedmuscle (Lehma net al., 1995) and it therefore actsas a relaybetweenthetroponincomplexandactin(Potteret al.,1985).Followin g anaction potenti al, Ca2+isreleasedfrom the sarco plasmic reticulum whichincreasesthe free Ca2+in the cytoplasm.TheseCa2+ions bindtothe specificsites on troponin C (TnC) which causes a conformationalchange on theTnC.This confo rmationalchange leadstoincreasedbindin g ofTnC to troponin I(Tnl)(relayedby troponin T), whichresults inadecreasein bindingbetweenTnlandactin. Therefore, thereis adecr easein theinhibitionthat Tnl hason the actomyos in MgATP ase (Zotand Potter,1987, review;Farah and Reinach,1995,review;Perr y1996,review;Lehm an and Craig,2008, review ).Itisbelievedthat thebindingofcalcium ionsto thetroponin comp lexshifts thetropom yosin onactin, thereb y expos ing themyosinbind ing sitesof actin(Vibertet al., 1997).
Using interf erencemicro scop yHuxley and Niedergerke showe dthat during shorteningorstretching thelength ofthe A-bandsremai nedconstantwhile that ofthe1- bands changed (Huxleyand Niedergerke,1954 ).Phase contrastexamination revealedthat theadditionof ATP tomyofibril sinorder to causecontraction result ed inthe simultaneo usshortening of theI-band and the H-zone (Hux ley and Hanson,(954). These result sled to the prop osal of the sliding filament theory(Figure2)inwhic h thethick
Figure 2: Diagram of theslid ing filament theor yof musclecontraction
(myosin)andthin(actin and tropomyosin)filamentmove pasteach other. In thistheory, myosinundergoes aconformational changeso thatit detach es and reattachestoactin. In theabsence of ATP, the myosincross-bridgeis stronglyattached toactin. Thebinding of AT P cause sthedissociationof actomyosin and theATP ishydrolyzedtoADP and Pi which'cocks' themyosinhead. Thishydrolysisinitiates a weak associationof the myosinheadtoactin and isfollowedbythe releaseof Piwhich is associated withthe power stroke . Dissoci ationof ADP thenprepares for another contract ion (Huxleyand Simmons,1971 ;Huxley1974;Lymnand Taylor,1971).
1.3 Maj orSar comer ic Proteins
The maj orprotein sin thesarcomereare myosin,actin, troponinC,troponinI, troponinTand tropomyosin .Although not discussedbelow ,musclealso containsahost of other proteinswhich arerequired for various aspects offilamentstructureincluding, anchoring(ega-actinin ), end'capping '(e.g. tropomodulin and CapZ )andspatial organiza tion(e.g. titinand nebulin) as wellas enzymes forthe produ ction of ATPand creatine phosph ate.
1.3.1 Myosin
Myosin, an enzymethathydrolyzes AT P(Enge lhardtand Ljubim owa,1939), accounts for greater than 50%of thetotalproteinin the musclemyofibril (Furukawaet al.,1972).Inthis tissueit isa hexameric protein containing a number of functionaland struc tural domains.Sedim entat ion experiments first suggested thecorrectmolecular weight of 470,000Da (Loweyand Cohen,1962). Later research showe d thatthere are
7
twoheavy cha ins(Mr- 230,000 Oa) (Gershma netal.,1969; Loweyet al.,1969),two regulatory light chai ns(M,-16,000-20,000Oa)(Lowey and Risby,1971;Weedsand Lowey,1971) and two essential light chains(M,-16,000-20,000Oa) (Loweyand Risby,1971;Weed s and Lowey,1971).Thesixchainsarearranged toformtwo globular heads attachedto ana-helicalcoiledcoil tail (Slayte randLowey,1967).The C-termi nal portion oftheheavy chains ismainl y a-helical and form sthemyosintail. The N-terminal part oftheheavy chains formstheheads.Eachglobular head also has oneessentialand one regul atorylight chai nassociatedwithit,andcontainstheAT P,actinand divalent cation-bindingsitesand is thesite of ATP hydrol ysis (Gaz ithelal.,1970). Crysta lx-ray diffractionrevealsthat thehead is approxi mately50%a-helica linstruc ture(Rayme ntet al.,1993). Thea-he lical tailshavethetypical coiledcoil heptapept iderepeatthat is describedlaterin moredetailinsection I.3.4(Crick,1953).
Myosinmolecules aggregate toform abipolarfilament structurewhich makesup the thick filam ent (Hux ley, 1963) (Figure 10).Loweyelal.(1969)hypoth esizedthatthe thick filam entisform edbecausemost of themyosintail iswaterinsolubl ewhilethe remai nderofthetail and the globular heads are water soluble.The struc tura lrole ofthe inso luble myosintail appears tobetheorganization of thethick filament (Loweyelal., 1969).Thispacking arrangeme nt result sin themiddleof thethick regionbeing ahead free region andaregionwhereactin-myosin crossbridges cannot be formed(Harfo rdand Squire, 1986).Importantl y,the globular headsproj ect outfrom thebody of thefilament (Figure 10).The organization of the myosin chains hastwo implications:I)themyosin
polarityis reversed ineach hal f ofthefilam ents and2)the ATPaseandactin binding sites areon theexterior of the filament(Aidley,1991p265-269, review ).
1.3.2 Actin
Actinwasaccidentally discoveredinSzeged,Hungaryin 1942byF.B.Straub (1942).Itis apervasiveprotein whic h isfound in nearl y alleukaryo ticcellsand isthe main componentof thethin filament (Ka bschand Vandekerckh ove,1992,review).
Musc leactin can existas eitheramonom er (G-actin)or as afilam entou spolymer (F- actin). G-actin,Mr-42,000Da,consistsof 375amino acid s(Elzingaet al., 1973) and thesequence hasbeenhighl y conserve d throughout evolution. Sequ encin g of cyanoge n bromidepeptid esrevealedthatresidu e 73isa 3-me thylhistidine(Elzinga, 1971).Itis believed that thisaminoacidisincorporatedintotheproteinby enzyma tica llyadding the methyl groupsat residue 73 with S-adenosylmethi onine actingasthemeth yl group donor (Asatoo randArms trong, 1967).Thefirstthreecodonsin the skeletal muscle actingene code for Met-X-Asp(X isusually Cysbut itcan beGly orAla)whereas themature proteinterminat eswithan acetyl-Asp (Zakutetal.,1982).Research on theDrosophil a melanogaste ractinsuggestsapossiblemechanismforremovingtheinitialtwo N- terminal aminoacidsofactin: the methionineisrem ovedbytheribosom al amino peptid ase,followedby acetylationofthecysteine ,rem oval of the acetylatedcysteineand finally the acetylationof the aspartic acidresidu e (RubensteinandMartin,1983).
Kabschet al.(1990) firstdetermin edthe atomicstruc ture ofG-actinincomplex with DNaseIat 2.8and 3.0Aresolutionsin thepresence of eithe rATPorADP.In the struc ture, thenucleotide was situated inacleftbetweena small andlargedom ain .The
9
small domain consistsof residu es1-144and 338-375 and thelargedom ainismadeupof residue 145-337. Each dom ain can be further dividedinto two subdo mains.Thesmall dom ainis comprise dof subdomainI(residues 1-32, 70-144 and338-375)andsubdomain 2 (residues33-69).Thelarge domainencompassessubdomai n3 (residues 145- 180 and 270-337)andsubdomain 4(residues 181-269).Theydetermin edthat subdomain I containsa five-strandcdBvsheetsurro unded by fivehelices andsubdo main2 consistsofa lhree-stranded antiparallel~-pleated sheetwith ahelix connectingthetwo edge-s trands.
Subdomai n3 is a five-stra nded~-sheetsurroundedbythreehelices and subdomain4 containsatwo-stranded anitparallel~-sheet andfour a-he lices.
Monomericaetin(G-actin)can aggrega te toform afilament ous polymer(F-actin) inthe presence of Ca2+and Mg2+and higherionic strengths.Upon polym erization,the boun dmolecul e of ATPishydrol yzedtoleavebound ADP(Szent-Gyorgyi, 195 1).
Murakamiet al.(2010)recently proposed amodeldetailingtheform ation of filamentous acting and thehydrolysis of ATP.They hypothesizedthat anewly inco rpora tedactin monomer enables the rotationofthe outer domain of thepenult imate actinmolecu le.An interactionbetween this actinandthe neighbo uring actinorientsthesubdomai n2of the formersothatitsDNaseI loopreaches outwa rd. Part of this loopfits intoahydroph obic cleft whileanot her part causesadownward shiftto aproline-richloop.Thisshifttriggers ATP hydrolysis and Piis release d.F-actinisthe forminvolved incontractionand itis compose dof twostrands thatarearranged ina do uble helix.F-actin,asdetermined by electro n microscop y,ishelicalwith13 actin molecul esper 6turns anda repealof approxima tely360A(Holmeset al., 1990).In the non-muscle system,thepolymerizatio n
of actinisdynami c and themonomersattachand detach from the endsof thefilament at differentrates (Wegne randEnge l, 1975).The endsare labeled asthebarbed (Plus)endor thepoint ed (minus)end. Actinmonomerswill bindateitherend, but it hasa preferenceto attac hat thebarb ed endand todetach atthepoint ed end(Woo drumet al., 1975).
Forma tionofF-actinstimulates the actinAT Pasebut the releaseofphosph ate is slower thanthe form ationof thefilamentresultinginanATP-actincapatthebarb ed end.
Monomericactincontai ning ADPand phosphate accumulate in therest ofthe filament (CarlierM-F, 199 1,review) .
Warm- bloodedvertebra tes contain sixactinisoformsthataretissue specific:two striated muscl e actins(cardiacandskeletal), twosmoo th muscle actins(vascularand visceral)and twonon-mu scle actins (pandy)inallnon-mu scle cells(Kabsc hand Vandeke rckhove, 1992, rev iew).
1.3.3 Troponin
Tropon in,discoveredin1965 (Ebashiand Kodam a,1965), is a complexof three regulatory protein s (Ebas hietal., 1968).Themolecularmasses of thethreetroponin subunitswere first determined fromthe aminoacidsequencesoftheprotein s obtained from rabb it ske letal muscle:troponin C (TnC, M,=18,000Da) (Collinsetal., 1977), troponinT(TnT, M,=30,5 00 Da) (et al., 1977a) and tropon inI(TnI, M,=21,000Da) (Wilkinso nandGrand, 1975).TnCisthe calcium binding subunit,TnT binds to tropomyos inand TnIisthe inhibitorysubunit(Grease randGerge ly, 1971,1973).The
atomicstruc tureofmost ofthe complex, the so-calledcore,hasbeenreportedinboth Ca2+_boundandfree froms (Takedaetal.,2003;Viongradovaetal.,2005).
1.3.3.1 Troponi nC
Trop on in C is thesmallestof thethreetropon in subunitscontainingabout 160 residu es andhas apI of4.1-4.4 (Hartshom e andDreizen ,1973).Itis adumbbell-shaped protein withalong centra l helix connecting the aminoandcarboxy -term inal domains (Sundaralingametal., 1985).Greater than 65%of the am inoacidsare in a-helical conformation(Herzbergand James,1985).Itutilizesthecommon helix-loop-h elix (HLI-I) Ca2+binding motif where a12residueloopis flankedbytwoa-helica lsegments(Collins etal., 1977).Troponin Cis capableof binding oneor two Ca2+ions,depend ing on the isotype,in low- affinit y Ca-specificsitesand two Ca2+ionsin high affinitysites(which canalso bind Mg2+ion s) (PotterandGergely, 1975).Thetwohigh- affin itybindin g sites (111and IV,K,-107M·I)are located attheC-termi nal(struct ura l dom ain) andthe low- affinity binding sites(IandII,K,_105-106M·I)are located attheN-terminal (regu latory dom ain) (Sundaralingametal., 1985;Pearlstoneetal.,1997).Itis thelatter whic hserve as thecaleiumsensor.
1.3.3.2 Troponin I
TroponinI isblockedby anN-acetyl groupand it has an isoelectri cpoint of 9.3.
The rabbit skeletalTnI iscomprisedof 179amino acids(W ilkinsonandGrand, 1975).It inhibits actomy osinATPaseand is capable of bindingtoactin, tropom yosin,TnCand TnT(Zotand Potter,1987,review).TheatomicstructureofTnI isnot yet known,
however, biochemica l,chemica landbiophysica linvestigations have shown that the protein can bedividedinto threeregions.The N-doma in(r-res id ues 1-95) interactswith the Cdomain ofTnCwhensites III and IVareoccupied by Ca2+(stron ginteraction ) or Mg2+(weakerinterac tion)(Farahetal.,1994).Thisintera ction ispartiall yresponsible for anchoring TnCto othermembers ofthetroponin complexand hasbothCa2+-dependent and-independent components(Pearlstone etal.,1997). Thesecond domain (r-residues 96-116)ismainl yrespon siblefor actomyosi n ATP ase inhibitoryactivity in the absenceof TnC and TnT.Residu e105-11 5 istheminimallengthrequir edfor inhibition to occur (Talbotand Hodges,1981).In the absenceofCa2+,it isbelievedthat thisregionisbound to actinand when expose d to Ca2+,it forms a complex theTnC (Pearlstoneetal.,1997).
Residu es117-156havea criticalregul atoryrole forthe Ca 2+depend ent interactionofthe third dom ain (r-residues 117-181)with the N domainof TnC(Fara hand Reinach,1995, review).
1.3.3.3 Troponin T
Trop oninTis acetylatedat its N-terminus(Pearlstoneetal., I977b)and themaj or isoformfound in therabbit skeletal muscleconsists of259 aminoacidsand has apIof 9.1 (Pearlston e et al .,1976 ;Pearlston e etal. ,1977b).Itis requir edfortheCa2+sensitiv e inhibi tionof ATPaseactivity(Grease randGerge ly, 1971).This subunit directlyinte racts withTnI, TnC and tropom yosin anditlinks thetroponin comp lex totropomyosin (Zot and Potter,1987,review).Also,TnT influencesthebinding of actin and tropom yosinin thepresenc e andabse nce ofCa2+(Heeleyetal.,1987).The N-term ina l dom ain of TnT interactswith trop omyosin (Jacksonet al.,1975;Mak andSmillie, 1981b;Pearlstone and
13
Smillie,1981;PearlstoneandSmillie,1982) whereas the globularC-terminal dom ain interacts with tropomyosin ,TnIand TnC (Ma lnicetal., 1998).
1.3.4 Tropomyosin
Tropomyosi nwas first isolated in 1946 (Bailey, 1946).Itisfoundinskeletal, cardiac andsmoo th muscle and(at lowerquantities)in non-mu scletissues (Smillie, 1979).Themuscletrop om yosin contains284aminoacidswhereasasthenon-mu scle form is sho rterwith approximately 247aminoacids(Smillie, 1979).Theproteinis a coiled-co il dimer (Crick, 1953;Cohenand Szent- Gyorgyi,1957 ;Woods,1967) and Smillie (1979)determ inedthat a subunitofa-tro pomyos infrom rabb it fastskeletal musclehas amolecul ar weight of33,000 Da.Sarco me ric tropom yosinhas adiameter of approx ima tely20Aand is approximately 400Along (I-litchcoc k-DeG regori,2008, review).Inamuscle,tropomy osinmolecules arearrangedinahead-to-t ailmann er along thelength ofthe actin filament(FigureIE).Itwasproposedthatthe N-andC-terminal endsof the284-res idue tropomyosin overlap by nineresidues(McLachlanandStewart, 1975). Amorerecent study ofan NMRstructureofthe overlap betweentropom yosin molecul esindicatesthatII residu es on theN-term inal ofone molecul efits into thecleft attheC-term inalofanother moleculethatis createdwhen thetwo strandsspread apart (Greenfieldetal.,2006).Astburyet al.(1948) showed thattropom yosin exhibiteda distinctperiodi citythat was characteristic of thek-m-e-f (keratin-myos in-e pidermi n- fibrinogen) groupofprotein s.Thediffractionpattern s oftropom yosin werearesult ofa left-handed coiled-co ilstruc ture involvin gtworight-h and ed a-he licesthatwere at a 20°
angleto eachother(Crick, 1953) (Figu re3C). Inorderfor thisto occur,a stripofnon- 14
B
Ii1l l!!J
Ste-r1M.ProcNOJIIAcOJd SCi98:196S.8166 Co p yrighl(200 1) Nl!llionai Acad etny o fSci ences, USA
c
Imagefromthe RCSBPDB (www.pdb.org) ofPDB10:ICI G
WhitbyFG,PhillipsJrGN (2000)Crystalstructureof tropom yosinat7Angstroms resolut ion.Proteins 38:49-59
Figure3: TheStructureof Tropomyosi n
(A) Amino acidpositions ofthe coiledcoil tropomyosin as viewedfrom an axial position.
Residues'a'and'd'are typicallyhydrophobic and helpkeep the twohelices in contact witheachother. Residu es's'and'e'are typicallycharged aminoacids that stabilizethe proteinthrough ionic interactions.Residues 'b'; 'c' and'f are usually charged.(8)Side viewinwhich the cylinders representthehelicalbackbone.(C) Molecularribbon diagram ofatropomyosindimer.
polar residuesrun downone side of each of the two a-helices.The helices windaround each other to formthecoiled-coilthatis stabilized by the "knobs andholes" packing arrangement (Crick,1953).In orderto create the non-polar sides of the helices, tropomyosin can bethought of ashaving aheptapeptide sequence(a bedefg ) where'a' and'd'arcnonpolaraminoacids.Residues'a'and'd'ononestrandarein contactwith residues'a'and'd'on the otherstrandandacidicandbasic residues arelocated onthe exteriorofthe coiledcoil(Figures3A and3B).Theremainingresid uesare usually polar orionicandsalt bridgesbetweenresidues'e'and's'oftenappea rinorder tofurther stabilize the coiled-co ilstructure(Mc LachlanandStewa rt, 1975).Johnson andSmillie ([975) have show n thatthetwo subunitsare in register andin para lIelduetothe fact that it ispossibleto formanintramo lecular disul fidebondbetweenthe cysteinesat position 190.
Tropomyosi n has anunbrokenseries of 40heptads thatis known to be unusual for fibrousproteins(Parry,[975). As well,additio nalstabilizatio nis addedto the protein becausethechargedresiduesinpositions 'e'and's'on neighbouringchains form salt bridgeswhich results in thetwo chains being in-register (McLachlanand Stewart,[976).
Most tropomyosi n molecules also have an unusuallyhighnumber of alanine resdiudes in thecore regioncompa red to othera-fibrous proteins.Invertebra teskeletal muscle,as manyas>35%ofthe'd'positionresidues are alanin es (Conwayand Parry,[990).
Brownet al.(200 I)describes sevenalanine clusters in thetropom yosinmolecule. These clusterswhichcause the chains topack close r togeth er (compared totherem aind er ofthe molecul e) arestagge redwhichresults inabend. The bendisimportant asitallowsthe
tropomyos in to windaround the actinfilament(Brownet al.,200I; SinghandHitchcock- DeGregori,2003).
Tropomyosi n ishighl y a-helicalwith the N-term inal hal f ofth emoleculehaving thegreatestamo untofhel ical content. Progressivelylesshel ical contentexists towards theC-te rminal(Smillieetal.,1980). Consistentwiththis,Greenfieldetat.(2003) showed that the N-te rminalformsa continuouscoiled-coil upto residue279 and that the last five residuesarein arand om confo rmation.Ithasbeen show n byPhillipsetat.(1980) and confirmed byBrownetat.(2001) thatthe N-term inalpart of tropomyos inismorerigid comparedtothe C-termi nal part.Lietat.(2002) determined thatthe C-termina lsectionof tropomyosin(resi dues254-284) doesnot form a coiledcoiland thatthetwohelices splay apartat thisendof the molecule. The siteof splay inginvo lvesa smallgroupof destabilizingcoreresidues includingGln263Cd')andTyr267Ca')that pack asymme trica llyat theinterface ofthehelices(Liet al.,2002).The mid-region of tropomyos in has threecorealaninesina row (residues 151-158) whic hcausesa 6°bend in the coiledcoiland there arealso isolated alanineswhic here ategapsalong theprotein (Brownet al.,2005).
More recently,Gly l26Cg)andAsp l37Cd'),both of whichare stro ngly conserved, havebeen show n to impart flexibility andinstabilitytothe middleregion of thetropomyosinmolecul e (Nevzorovet al.,20 II;Sumidaet al.,2008).
/.3.4./Tropomyosin/so/arms
Tropomyosi nispresent in virtuallyalleukaryoticcells.The forms ofthe protein which havebeen found innon-muscle tissues areshorterinlengthby one (platelet,Lewis etal.,1983)two (yeast Tpml,Liuand Bretscher,1989) or three (yeastTmp2,Dreeset al.,1995) actin binding sitescompared tothosein muscle, witha fewnotedexceptions (MacLeodetal.,1985).Thefirstevidence thatthere are multiple form s of tropomyosin camefromelectro phores isseparationsperformed in thepresence of SDS(Cumminsand Perry,1973).Two variantswere observed,so-calleda andp.The basis ofthe separation isnot due to atruedifferencein mass butnetchargewhic haffects the interactionwith SDS.Protein sequencingshowe d that 39 aminoacid differences exist between these isoform sin rabbitskeletal muscletropom yosin , with the C-terminal halfbeingmore divergent (Ma ketal.,1980).Further,p-tropomyos inispredictedtohave ahighernet negative chargeat neut ralpHthanadueto Ser229Gluand His276Asn (Ma ketal.,1980).
Thesequenceanalysisalsoconfirmedadifferenceincysteinecontent(Cumm insand Perr y,1974).Subse quently, fourtropom yosin geneswere discoveredin mamm als from whichat least 20isoform s areexpresse d by alternative prom oters and RNAprocessing (Pittengeretal.,1994).In humans,thefou r genes havebeenlabeled TPM I,TPM2, TPM3and TPM4(Cutticchiaand Pearson,1993).TPMIand TPM2 correspondtothe genes that areresponsible forthea- and p-trop omyosinslocatedinskeletal muscle (Helfmanetal., 1986;Ruiz-Op azo andGinard 1987).TPMIcontains 15 exons,and through alternativesplicing,cangive riseto at least eightotherisoforms including smooth muscl e a-tropomyos in.The p-tropo myosi ngeneconta insnineexons(fiveof
whichare inall isofonn s) andcodesforskeleta landsmoot h muscletrop omyosin and fibroblast trop om yosinisofonn s (Perry,2001, review). TheTPM3genecodesforthe 248 residuefibroblasttropomyosinanda 284 residueslow twitchskeletalmusclea- tropomyos in(TM3) (Claytonet al.,1988; Gunninget al., 1990;Smillie, 1996).The TPM4genecontainseightexonsandcodes for fibroblastTM4(Lees- Mille ret al.,1990;
Macleodetal., 1987).Itshould benotedthatthetropom yosin whichwas selectedfor study in thisthesis speci fically,salmo n fast ske leta l,is ofthe a-type,asdetermin edby sequencing(Hee leyet al.,1995).
1.3.4.2 Post-Tra nslationalModifications
Skeleta l muscletrop om yosinundergoestwopost-t ranslat ionalmodifications whichare:N-tenni nalacetylationand phosphor ylation ofserine283(HodgesandSmill ie, 1973; Maket al.,1978).The acetylationof the N-tenni nal meth ion ine isnecessary forthe tropomyosin tohave a stro ngbindingaffinity to actin(Urbancikovaand Hitchcock- DeGregori,1994). The absenceof acetylatio ninp-tropo myosi ncausesaneight-fo ld reductioninactinaffinityanditsabsenceina-tro pomyosin result s ina40-foIdreduction in actinaffinity(Coultonet al.,2006).Althoughacetylation isnotnecessary for homo- or heterodim erfonn ation, itappears to stabilize thecoiled-coil (Gree nfieldet al., 1994).
Covalently boundphosph atewasfirstdetectedin tropom yosinfromthe ske letal muscle ofafrogthat hadbeeninjectedwith32porthophosphate (Ribulowand Barany,1977).
Subsequently it hasbeenfoundin the striated muscletropom yosins ofrabbit ,chicken, fish,rat and mouse(Montarrasetal.,1981;Heeley and Hong,1994 ;Heeleyetal.,1982).
Onetheo ry forthe roleof phosphorylation oftropom yosin sugges tsthat it has 19
significance for myofibrillo genesis (Heeleyet al.,1982),while another proposesthatit plays a roleinstabilizing thehead-to-tailinteractionduringpolymerization (Ma kelal., 1978;Heeleyet al.,1989).Heeley (1994) demonstratedthattrop om yosin phosphorylation increases the steady-sta terateofhydrolysis of actomyos in MgATP ase.
Laterresearchusingphosphorylated and unphosph orylated shark tropom yosin confirme d this activa tion(Hayleyelal.,2008) .Itisimportantto notethatthephospho rylation is uniqueto striatedtropomyosinbecause smoothand non-mu scleisoform sdonothave a serineat position283 (Sandersand Smillie, 1985).
1.3.4.31I1t eract;olls w;th S e/faml otherProte;lIs
Tropom yosinisassociated with theI-band and thinfilam ent ofske letal muscle anditinteractswithactin, troponinT(Cors iand Perr y,1958;Perry andCorsi, 1958;
Eisenbergand Kiell y,1974).Tropomyosinlies along thenarrow grooveofthe actin helix (McLachlanandStewa rt, 1976).Onetropom yosinmolecule spansseven polymerized actin monom ers even thoughtrop om yosincont ains14 quasiequ ivalentrepeatingregions of acidicand non -polarresiduesthat couldserve asbinding sites(Stewartand McLachlan,1975).Ina tropomyosi n dimer,there are28 acidiczoneson the surface whicharearra nged in 14opposing pairstoproduce foursetsofsevenzoneswhichareat 90°to each other (Stewa rtand McLachlan,1975).Tropom yosinbinds cooperatively to actin, but theioni c conditions that are necessaryinvilro dependo n the spec ies(Yangel al.,1977;Yangetal., 1979; Lehrerand Morris,1982).Mg2+isimportantin formin gthe actin-tropomyos incomplexand it isthoughtthatMg2+bridgesform betweencarboxylate groupson bothprotein s (Ma rtonosi, 1962).Localinstabilit y and side cha in flexibilit y of
20
tropomyosinisnecessaryfor actinbinding(Singhand Hitchcock-DeGregori , 2006).
Singhand Hitchcock-DeGregori (2006)predicted thatinaddi tiontotheflexible coiled- coil,a spec ific recogniti on siteon the surfaceof thecoiled-co ilis alsoimportantfor actin binding.McLach lanandStewart(1976) first showe d thattrop om yosinhad seven periods that werequasi-equ ivalentfor actin binding. Further researchby SinghandHitchcock- DeGregori (2007) illustratedthattheseperiods alsohad specific regul atory functions.
Period 5 (res idues 166-207) was the most conserve d period and itwas also themost importantforbindingtofilam entous actin(Hitchcoc k-DeG rego rielal.,2002).Itis thoughtthatperiodIis also veryimportantfor maxim albinding affinity toactin because iti sthe onlyother periodthat containsalanineclustersembedded within theconsensus repeats(Singhand Hitchcock-D eGregori,2007).
TroponinT isresponsiblefortheinteraction ofthetrop onin comp lexwith tropomyosin (Zotand Potter,1987, review).Itis a stablecomplexthatforms independentl y of calcium butis sensitiveto theionicstrengthof itssurro undi ngs(van Eerd and Kawasaki,1973;Jacksonelal.,1975).TropomyosinandTnT interactwithaI:I molar ratio(Grease relal., 1972).Thereare twotropom yosin interac tionsitesonTnT.
Firstsiteislocated within the N-terminal halfof TnT, betweenresidu es 71-151 (numberingsystemcorresponding torabbit skeletal protein) and the secondsiteis within the C-terminal hal f ofTnT(resid ues 159-259) (Jackso net al.,1975; Ohtsuki, 1979).Itis genera lly believedthatthemajority ofthe C-terminalfragme ntofTnTisneededfor interactionwith tropomy osin (PearlstoneandSmill ie, 1981;Jacksonelal..1975).Site I
on TnT isthoughttobindtothe C-tenni nal part of trop om yosin whereassite2bindsnear Cys190 (Patoet al.,1981b;Chongand Hodges,1982,Morrisand Lehrer,1984» .
1.4 Atlantic Salmon Tropomyosin
Thebulkof thebiochem icaltropomyosinlitera ture described above ,pertainsto mamm alian andaviansourcesoftheprotein.Less work hasbeen carriedoutwith tropom yosinfromcold dwellin g organismssuchasfish.Despit ea widephylogenetic separationanda substantial differenc eingrowth temp erature,thenumber ofsubstitutions thatexist between tropom yosinsfrom fish and higher (warm-blooded) vertebrat esis small. For example,the sequenceof Atlanticsalmonfast myotom altropom yosin (below), inferredfromits cDNAsequence(accession numberNP_00 1117 128)(Heeleyet al., 1995),containsamere 20aminoacid replacem entscompared to rabbitalpha(accession number NM_ OOII05688) (StoneandSmillie, 1978).Yet NP_00 1117128hasbeen shown tovary significa ntlyfrom themamm alian versionin term s ofits confo rmat iona lstability (Goonasekara and Heeley,2008).Further,bothhalvesofsalmontropomyos inwerefound tobe ofcomparative ly lower stability(GoonasekaraandHeeley,2008).
rndaikkkmqrn lkldkenald raegaegdkk aaedkskqle ddlvalqkkl kgtedeldky seslkdaqek levaektatd aeadvaslnr riql veeeld raqerlatal tkleeaekaa desergrnkvi enraskdeek rnelqdiqlke akhiaeeadrkyeevarklv iiesdlerteeraelsegkc seleeelktv tnnlksleaqaekysqked kyeeei kv l td klke a e t r a e faersvakle ktiddledel yaqklkykaiseeldnalnd rnt s i
Ofthe 20 substitutions (TableI), onlyone affects a core amino acid specifically, residue-l79 whichis a threoninein salmonand an alanine in rabbit. In the amino-terminal half, there is a unique pair of glycinesat positions 24 and 27. These substitutions were viewed as potentially important determinants of the unique properties of salmon tropomyosin.
1.5Sites of ProteolyticDigestion in Tropomyosin
Throughoutthis studyOmp-T andchymo trypsinwere usedto studythedigest ion patternsof tro pomyos in.Patoetal.(l98 la)examinedthedigestionpatternsof rabbit striated a-tropomyosinby limited chymot rypsi nproteolysis .They showed chymotrypsin initially cleaved tropomyos inonthe C-terminal side of Leu-169. Thisresidueis in the core of the protein and was believed to be fairlyinaccessibleto theenzyme.The researchersproposed that this residue is in a destabilized area due to the presence of several bulky hydrophobic residues at positions 169-172 (-Leu- Val-Ile-Ile-). They also speculated that an aspartic acid (AspI75) ina'g ' position results in an electrostatic repulsionwith Glul80whichcontributes to that region's instability.
The Omp-T digestion patterns of muscle tropomyosin were first explored by Goonasekaraet al.in 2007.Edman sequencingwas usedto prove that Omp-Tcleaved betweenLys6 andLys7 onsalmonskeletaltropomyosi n.The researchers showe dthatthe cleavageoccurre dat the samesite on tropomyosin from several differentsources includi ngsharkskeleta l trun kmuscle,bovineheart, chickenbreast,chicke ngizzardand rabbitskeletal muscle.Goonasekaraetal.(2007)alsodemonstrated that salmonskeleta l
23
Table1: Differencesbetween the rabbit and salmon fast muscle tropomyosin
Position Heptapeptide Salmon Rabbit
desiznation
~4..}.~Lk~~c Gly GIn
:,~1,~f Gly Ala
35 g Lys Arg
42 g Asp Glu
45 c Ala Ser
63 g Ser Ala
73 c Val Leu
77 g Thr Lys
111 f Thr GIn
132 f Asn Ser
135 b Ser GIn
143 c Leu lie
145 e Asp Glu
157 c Glu Asp
r![~~'i?fIA~.d Thr Ala
191 b Ser Ala
229 e Thr Ser
247 b Ala Thr
252 g Thr Ser
276 c Asn His
Shadedboxesdenote substitutionsof interest that weremutatedin the curre nt research.
24
muscletropom yosinismore susce ptible todigestionthantherabb itcounterpartand that theunacetyl ated salmon tropomyosiniscompletelydigestedat 30 min whereasthe N- acetylatedsalmon tropomyosinwasmainlyintactat thistime.
1.6 Psychr ophilicProteins
Organism s can be placed in three catego ries basedon the surrounding temp erature thattheythrive on.Mesophile sthri vein temperatures of17-40°C,therrnophil esthrive at temp eratur esofgreater than 50°C, and psychrophil esthriveat temp eratur esbelow15°C (Gerdayet al.,1997;Greavesand Warwicker, 2007).Mostofthe researchthat hasbeen carriedouton protein sfrom psychrophilic organisms hasbeenfrom the standpo intof globular protein s.It hasbeen shown,ina given famil y ofglobular protein s,that compared tomesophil esand therrnophil es,psychr ophil eshaveincreased loop sizes and decreasednumbers of ion pairs,hydrogenbonds and hydrophobi c contacts (Greavesand Warwicker2009;Greavesand Warwicker2007;Gianeseet al., 2002). Thesedifferences provide psychrophilicproteinswith the necessaryflexibilityto functionat low temp erature.They alsoaccount forthefactthat such proteins aregenerally unstableat mesophili ctemp eratur es and higher.
Spec ificam inoacid differences oftenoccurwhencomparing psyc hro philes to therrnophil es: arginineand glutamin e are repl acedbylysin e andalanineonexposeda- helic es sites, valineisreplaced by alanineat buried regions on a-helic es,there isa significant increasein alanineandasparagine atexpose dsitesandadecreasein arginin e and the buried sitesofBvstrandsare depletedofvaline(Gianeseet al.,2 00 [). Some
research hasbeendone on the stabilityofthenon-globul arproteintrop om yosin which confirms thatthe core position aminoacids('a' and'd)arecritica l to itsstability.
Hayleyet al.(2008)reportedthat shark tropomyosin (psyc hro phile) hasthree coreamino acidsubstitutions comp aredtorabbittropomyosin (Stoneand Smillie,1978).Ineach case,the equiva lent amino acid isreplacedby ahydroxyl-cont aining one,with the associated loss of twoalanines:Thrl79Ala,Serl 90Cys andSer2 11AJa.Examinationof sharkand rabbittrop om yosinindicatesthatthe shark isof ormhas aredu ced conformational stability throughmostofitsstructure comparedtotherabbit isoform whichisaresultof the core amino acid substitutions (I-1ayleyet al.,20 11).
1.7MutantTropomyosins
Four mutant s ofsalmo nfast muscletropom yosin were usedthroughoutthis researchproject-three single mutations andone doubl emutation (Ta ble I). Each mutant invo lvedthesite-directed mutagenesis of acore aminoacidandwas selectedfora specific reason.Three of themutant s (Gly24A la,Gly27A laandGly24A lalGly27A la) werechose n because of theuniquedoubl e glycines that arepresent attheN-terminalof the salmo n tropom yosin (Jackmanetal., 1996).Glycin eresidu es are morecomm on on the C-termina l hal f of a-helicesand in loops,(Richardso nand Richard son,1988).
Researchindicatesthat glycineallows for a moreflexibleprotein compared to alanines becausetheformercan ado pt morephiand psi ang les(Creighton, 1993;Serran oetal., (992).Thesalmo n tropomyo sin glycines weremutatedtoalanin esfora couple of reasons.Primarily,alanine was chosen because it isthe standardreplace me nt. Also,in
rabbit ske letal tropom yosinthereis analanin e at position 27 instead ofa glycin e.All threeglycin emutant sweremadetoexamine both theindividual andadditive effect of thatresidueon the conformational stability.
Thethreoninemutationwas of interest becauseit isthe only substitution(salmon vs.rabbita)that occursatacorepositionon thetropom yosinmolecul e (Hee leyet al., 1995).Itis also importanttonotethatthisisom orphi smplaces apolar side chainin a regionof knowninstabilit y (Lehrer, 1978). As aresult of havinga threonin ein position 179,thercarcthreeconsecutive'd ' am inoacidsthat containdestabili zin gamino acids (lieat 172and Serat 186). Thisthreonineoccursinaregion (172- 192)of theproteinthat isknowntobe susce ptible to familial hypertrophiccardiom yopath y causing mutations (Thierfelderet al.,1994;Golit sinaet al.,1997;Binget al., 1997).Thethreon ineresidue wasmutatedtoanalanine because alanine istheresidu ethatispresent at the same positi oninrabbitskeletal tropomyosin (Stone and Smillie,1978).
1.8 Goalsof Study
Themain goal of this study wasto characteri zetheinfluencethatparticular amino acidsinsalmo nfast muscletropomyosinhave on theprotein ' sflexib ility.Thethree residu esthat were ofinterest were Gly24,Gly27 and Thrl79.Itwashypoth esizedthatthe ThrI79 increases flexib ilitybecause itis ahydroph ilic aminoacid located in a predomin antlyhydrophobi ccoreregion of theprotein. Theglyc ines werehypothesizedto enhance flexibilityowing to thehydrogen side-chain which allowsfor a greaterrange of phiand psi angles.
Theeffect that these resi dues haveon theconformat ionals tabilityofsalmon tropomyosi nwas inves tigated by charac terisi ng theconsequence of mutating a given residue to alanine usingheat-indu cedunfolding inconjunct ionwithcircular dichroism and limitedproteolysisinconjunctionwithEdman-basedsequenc ing.Thefindings describedherein offerinsight intothe factorswhich modul ate conformationalstabilityof rod-shaped protein sin psychrophil es,whichhavenotbeen studied tothe sameextentas globular proteins.
The specific goalsareenumerated below :
I)Use site directedmutagenesisto create fourmutant salmon fast ske leta l tropom yosin :Gly24Ala,Gly27Al a, Gly24A Ia/Gly27A la,Thrl79Ala 2)Isolate enr ichedproteins sampl esusing salt-precipit ation andion -exchange and
hydroxyapatite chro matography
3) Use a circular dichroism spectrophotome ter tomon itor the heat-indu cedunfolding of the fourmutant and thenon-mut ant recombinanttropomyosi ns inorderto determ inetheirmeltin g temperatures
4) UseOrnp-T digestionto compare the ratesofN-te rminalcleavage of the non- mutanttropom yosin, Gly24A la,Gly27AlaandGly24A la/G1y27Alaat 10, 25 and37°C
5) Usechymo tryps in digestionto comparethecleavage pattern s of thenon-mut ant and Thrl79Alatropom yosin
6)Sequence electrobl otted fragments in ordertodetermin ethe initials iteo f chymo trypsin cleavage
Chapter2 MaterialandMethods
2.1 pH Measure ments
Arefill able combinationcalomelglasselectrode(Futura™,12 x 130mm, Beckma n)attac hed to aBeckman11J32 pH meter was usedfor all pHmeasurements.The electrodewas calibra ted dail yusing standardsolutionsofpH 7.00 andpH4.00.
2.2 Electro phores is
SDSPAGE was perform ed according to the methodsdescrib edbyLaemmli (1970) using12%(w/v)acrylamide(BioRad) diluted froma stocksolution(30%w/v acry1amideand0.8%bis acrylamide)with separat ingbuffer(0.75M Tris-HC I,0.21%
w/vSDS, pH8.8) (totalvolumeof20 ml). Polymerization was achievedwiththeaddition of17.5ulN, N, N',N'-tetrameth yleth ylenediam inutese (TEME D: Promega) and 120).l1 of a10%(w/v)solution of ammonium persul fate (APS:Prom ega).Stack ing gelswere dilutedto 3%(w/v)fromthe samestocksolutions(totalvolumeof5.5 ml) and polymerized with80).l1of APSand6).l1ofTEME D.ABioRadProtean" appara tuswas used for electrop horesis .Gelswere generally7.0ern long,10.0 ernwideand0.75 mm thick.Allsampleswere dissolved in SDSsamplebuffer (bromop heno lblue,glycerol, SDS and DTT). Electrophoresiswas carriedoutata voltagebetween 120-l 80Vuntil the bromoph enolbluedyeran offthe gel.Gelswerestained in0.25%(w/v)Coomass ie Brilli antBlueR-250 (BioRad),50%(v/v) ethanol (or meth anol ) and 10%(v/v)aceticacid and thendestained in 15%(v/v)aceticacidand20%(v/v)ethanol(or meth anol). A
volume of 10IIIof Precision PlusProtein Unstained Standard s (Bio Rad,cat#161-0363, 10-250kDa) wereusedtoestimate themolecularweightof theprotein.
2.3Dialysis
Dialysis wasperform edusing either narrow(10mmwidth;12,000- 14,000Da MWC O)orwide(50 mmwidth;6,000 -8,000 DaMWCO)dialysistubing (Spectrum, California). Dialysistubingwaspreparedbyheatingto-70°Cin-1.8Lof10 mM sodium bicarbonat eandImMEDTA. Thiswasfollowedbyextensivewashing (3-4 changes) of deionizedwater(dH20).The tubingwaslefttosoak overni ghtin dH20 and wasthen storedat-4°Cin70%(v/v)ethanol. Priortouse,thedialysistubing was thoroughl y washedwith dH20.
2.4 Spectroscopy
2.4. 1UV-VisibleAbsorbance
ProteinconcentrationsweredeterminedusingUV absorb ancemeasurementsin eitheraBeckm anDU-64 spec trophotometeroranAgilent8453 Diode Array spectrophotome ter. Tropom yosin was dialyzed(narrow dialysistubing,12,000 -14,000 Da MWC O)overnightat - 10°C againsta givenbuffer and theundissolvedprotein was rem ovedby centrifugationat 12,000rpm(Eppendo rf micro centri fuge 54 15D, F45-24-1 1 rotor) for 2min. For the Beckman spectrophotometer, theinstrum ent was calibrated using dH20and theabsorbanceof boththedialysisbufferand theproteinsamplewasmeasured at relevantwavelength s.Measurement s were between0.1-1.0absorb anceunits. For the
DiodeArrayspectrophotometer,theinstrument wascalibrated withair,and the absorbanceof dH20,dialysisbufferand proteinwere measur ed at relevantwavelengths.
Anextinctioncoeffic ient,E280I mglml,ofO.25 wasused after corr ection forscatter by subtracting1.5xA32o(l ohnsonand Taylor,1978).Themolar massof tropomy osin was takentobe66,000 glmole.
2.4.2Circular Dichroism
Approximately10 mg of freeze-driedtropomyosin(eithermutated or non- mutated) was dissolvedin2mlof buffer (0.1M KCl,20 mM potassium phosp hate, 0.01%Na azide,ImMEGTA, 1.5 mM OTT,pH 7.0)and dialyzed(narrowdialysis, 12,000-14,000 Da MWCO) overnight againstI L of thebuffer in thecoldroom.
Follo wingdialysis,the sample wascentrifugedto remove any insolubl eprotein.Aprotein concentrationof eitherI or 2 mglml sample wasused .Spectrawererecord edusing a Jasco 810spectropolarimeter. Mutant and non-mut anttropomyosinswereheatedfrom 5
°cto 65°C in jacketedcells ofvarying lightpath(0.1 mm or 0.2 mm)whilst simultaneouslymeasurin gthe ellipticityat 222nm.Thetemp eratur e was contro lled by a CTC -345 circul atin gwaterbath.Thetemperatur eincrease wasperform ed atarate of either 30or60°C/h r. Thescanningspeed of theinstrum entwas setat 100 nm/minwith normal sensitivity. The meltingtemperaturewasobtainedbydeterminingthetemperature of the normalizeddata at 50%unfolding.
2.4.3 Nanodrop
A Thermo ScientificNanodrop 2000 machinewasusedto determinethe concentrati onof DNA.The machinewasfirstblanked using2 IIIof nucleasefree water and then the absorbance of the sample was measured.Absorbances weremeasured at260 and 280 nm and the ratio of the twoabsorbance measurementsindicated the nucleic acid purityof the sample.
2.5Site-directed mutagenesis 2.5. / Oligonu cleotidePrimers
Custom-madeoligonucleotide primers were obtainedfrom Operon(Huntsv ille, Alabama).All primers had a melting temperature between73and 75°C.The underlined nucleotide is the one that differs from the original cDNA. The following primerswere used for the site-directed mutagenesis:
Mutation:Gly24Ala 5' TGGACAGAGCTGAGGf AGCCGAGGGAGACAAGA3' FORWARD 5' TCT TGTCT CCC TCGGCT~CC T CAGC TCT GTCCA3'REVERSE
Mutation:Gly27Ala 5' GCTGAGGGAGCCGAGGf AGACAAGAAGGCAG 3' FORWARD 5' CTGCCTTCT TGTCT~CCTCGGCTCCCTCAGC3' REVERSE Mutation: 5' GCTGAGGCAGCCGAGGf AGACAAGAAGGCAG 3' FORWAR D Gly24Al a/Gly27Ala 5' CTGCC TTC T TGTCT~CCTCGGCTGCC TCAGC3' REVERSE Mutation:Thr179Ala 5'AGTGA TCTGGAACGTGf AGAGGAGCGCGCTGAG3' FORWARD
5'CTCAGCGCGCTCCTCT~CACGTTCCAGATCACT3' REVERSE
2.5.2 Purification ofpTRC99APlasmid from BL2] Cells
In thissection,allcentrifuga tionstepswere performe d in an Eppendorf microcentrifuge5415D,F45-24-11 rotor atroomtemperature (RT).BL21 cells(60~I) with the expressio nvector pTrc99A (Pharmacia,27-5007-0 1),whichcontains the AtlanticSalmo nfastske letal muscletropom yosin cDNA,(Jackma net al.,1996) was usedtoinocul ate 6mlof LB broth containing25ug/ml chloram phenico land I00ug/ml ampici llinwasincub ated overnightat37°Cwithagitat ion. TheDNAfromthe overnight culture waspurifiedusingtheWizardplus miniprepkit(Promega)inaccordancewith manuf actur er'sinstruction s.Thecells weresedimentedat 12,000rpm for 2min and resuspend edinJuo ulofcellresuspension solution.Cell lysis solution(300 ~ 1)was addedand the sampleswereinvertedseveral times.Neutr alization solution(300~I)was added to and the samplewas againinve rtedseveral times.Sampleswere pelleted by centrifugat ionat15,000rpm for10min and thesupernatant waspurifiedusingthe resin providedinthe kit.Themini-column was washed using the wash solutio n provided followe dby centrifugatio nat 12,000rpm for 2min.50~Iofnuclease-freeH20was used to elute theDNA followe d by a shortcentrifugatio nat 12,000rpm.
2.5.3 Site Directed Mutagenesis
Site-direc ted mutagenesis was carriedout bymean s of thepolymerase chai n reactionusingtheQuikCh angeLightnin gSite-Dir ectedMutagenesiskit(Stratagene) .All reagent s,unless otherwisestated,wereprovidedin thekit. Double-strand edDNA templ ate(10-100ng),containing the Atlantic Salmon trop om yosin cDNA (Jackm anet al.,1996)wasmixed with5~1of lOXreactionbuffer,1~Iof dNT P mix,1.5~Iof
QuikSo lution reagent and 125ng of eachofthetwo custom- madeoligonucleoti de primers(Ope ron).Thesamplewasdilutedto afinal volumeof 50 IIIwithsterile dH20 andI IIIof QuikC hange Lightnin g enzymewas added . DNA was amplified using a MJ ResearchPelt ier Therma lCycler200 machineusing thefollow ing parameters:
Segment Cvcles Temo eratur e Time
I I 95°C 2min
2 18 95°C 20sec
60°C 10sec
68°C 2.5min
3 1 68°C 5 min
Following amplification ,thereactionmixturewasdigestedby additionof2IIIofDpn1 restri ction enzyme and incub ation at37"C for5min.
2.5.4 Transformation ofDNAwith XLIO-Gold UltracompetentCells
p-merca ptoe thanol(2Ill) was added to 45IIIofXLlO-Go ldultraco mpetentcells inapre-chill ed tubeandincubatedonice for2min. 2III ofDpn 1 treatedreactionmixture was added to thecellsandincubatedonicefor 30min.After30min,itwasheatpulsed in a 42°Cwater bath for30 sec followedby incubationonice for 2min.0.5 mlofNZY+
broth (prehea ted ina water bathto 42°C)was addedtothe reactionmixtureand incubatedat37°Cfor1 hrwithshaki ng.100 and200IIIof thetransformat ionreaction wasplated ontoLBagar platescontaining251lglmlchloramphenico land 100ug/ml ampici llin.Theplateswere incubatedovernightat 37°C.Follow ingovernight incubation , a colony from theplatewasusedtoinoculate 5mlofLB broth containing 251lg/ml chloramph enic ol and 100 ug/mlampicillin. The culturewasincub ated overnight at37°Cwithshaking .
1.5.5 TransformationofDNAwith BL1I Cells
450 ulof O.IM CaCI2was added to50 ul of compet entBL21cellsin pre-chill ed tubes.10-100 ngof DNA(purifiedfrom XL IO-Goldultra competent cells,usingthe same procedur eforpurification of DNAfromBL21 cells) was addedand the samplewas incubatedon icefor 30min;The samplewasheat shockedfor60 sec at 42°C and incuba tedon icefor 2min.900filofpreheated (42°C)SuperOptimalbroth with Cataboliterepress ion(SOC) medium (Invitroge n) was addedand thetransformati on reactionwasincubat edat 37°CforI hr withshaking. Thecellswereconc entratedby centrifugationat 12,000rpm for2min and the entire transformati onreaction was spread ontoLBagar plates containing25fig/mlchloramphenico land 100 ug/mlampicillin.The plates wereincub ated overnightat37°C.A whitecolonywasusedto inoc ula te 10mlof LB broth containing25ug/rnl chloramphenico land 100 ug/m lampicillin.The cell culturewas incubat edat37°Covernightwithcontinuous agitation.The cellculturewas storedat-8 0°Cin equi-volum e glycerol.
2.6 DNA Sequencing
Mutation s wereconfirmed byDNA sequencingatCREA IT(Me moria lUnive rsity of Newfoundland,NL,Canada) .Theprime rs(from Operon )usedto sequence the tropom yosin inse rtoftheexpress ionvector were:
M13/pUC reverse 5' AGCGGATAACAAT TTCACACAGG 3' pBa d -rev 5' ATCA GACCGCT TC TGCGT TC 3'
CREA IT uses anApplied Biosystems ABI3730x Isequence rthatis automatic except for some prelimin ary set-up.Foreachsample,2ul of5x sequencing buffer, 0.5ul ofsequencing mix,3.2pmol ofa primer,DNA template(at least500 pmol) and nuclease- freewater to obtaina volumeof20~Iwas addedat RT.Samplesweremixedby vortexi ng briefl y ina table topmicrocentr ifuge.pGEMwas set upas a control.A 9800 thcrmocycler (Applied Biosystem s) wasused for PCR amplificatio n usingthe follow ing program:sixminutes at96°C,25 cyclesat96°c for10 secondseach,50°Cforfive seconds,60°Cforfourminutes and then4°Cuntil the samples wererem ovedfromthe therm ocycler.The samples wereagainbriefly centrifuge dat RT.
The Agenco urtCleanSE Qsystem(Agencourt Bioscience)wasusedto purify the PCR product for sequencing . Briefl y,10~Iof AgencourtCleanSE Q magnet ic beadswas added to eachsamplefollowed by 62~Iof85%ethano l(vol/vol)and mixed thoro ugh ly.
Sampleswereplaced ontoamagneticplate (AgencourtSPRIPlate69R).Whenthe solutionwas clear(3-5 min),it was aspirated fromthe sampleand discard ed.A100~I aliquotof 85%ethanol(vol/vol)wasaddedtothe sampleandafter30seconds, the ethanolwasdiscard ed.This ethanolwashwasperform ed againand the sample leftto air dry for 10min.Deoinized water(40~I)was added tothe sampleafter whic h itwas removed fromthe magnetic tray.After5min,the samp lewasreturn ed to the magnetic trayto isolate theDNA. After5min, 35~Iof samplewasloaded intotheABI3730xlfor sequencing.Sequencing result s wereanalyzed using FinchTV from Geospizaand compared tothe cDNA of AtlanticSalmonfastmuscletropom yosin (Accession# NM_001123656).
2.7Express ionof TropomyosininBacteria
In this section, unless otherwisestated, centri fugation wasperform edina Beckm anJ6-H C centrifuge at 4°C.Bacterialexpr ession of therecombin anttropom yosin (mutatedand non-mut ated) wasperform ed asdescribedbyJackm anet al.(1996).Briefly, 40 mlof LB broth conta ining25ug/mlchloramphenico land 100ug/ml ampicillinwas inocul ated with400 J.lIofcellscontaining themutatedtrop om yosinDNA.Thecell culturewas incub ated overnightat37°Cwithagitation.4 Lof LB broth containing25 ug/mlchloramphenic ol and 100 ug/rnlampicillinwasinocul atedwith the40 mlof overnightcellculture.
The culturewasincubated at37°Cwithshak ingandtheabso rbanceat600nm wasmeasured.Whenthe abso rbancereached0.6-0.8,express ionoftropomyosin was indu cedusing 0.5mM isop ropyl P-D-thioga lactopyra nosi de(IPTG)(overnight,37°C, withshaking) . The followin gmorning,the cells werepelletedina centrifugeat 4,000 rpm for 20 minutes.Thecellsweredispersedin 100 mlof 0.2M NaCl, 50 mM MOPS,I mM OTT at pH 7.0 and passedthrough a French pressure cell at RT. Thelysate wasmixed with 1200mlofthe above buffer andstirred for15 min(RT) followin gthe additionof PMSF (saturated,in95%ethanol).Sampleswere centrifuge dat 4,000rpm for 20min.
Thesupernata ntwaspI precipit ated (4 .6)andcentrifuge dat 4,000rpmfor 30min.
Thepelletsweredisper sedin800 ml totalof buffer (0.2M NaCl,50 mM Tris, 0.5 mMEDTA, 0.25mM OTT pH 7.9)at-10°C for 15 min(w ith PMSF).Sampleswere centrifugedat 4,000rpm for 20 min inaBeckmanJ6-HC centrifuge. The supernatant
precipitated by ammonium sulfate (45%)followedby centrifugation at 4,000 rpm for 30 min in a Beckman 16-HC centrifuge. Centrifugationwas followed by 70%ammonium sulfateprecipitationof thesupernatant. The sample was then centrifugedat 8,000rpm for 45 min in a Beckman12-2 1centrifuge (JA-1O rotor) at 4DC.
Thepelletswere dissolvedindH20 anddialyzed(wide dialysis,6,000 - 8,000Da MWCO) against dH20 (containingammoni umbicarbona teandmercaptoethanol) at - 8 DC for 2 days with4 waterchanges(for atotal of20 L). Finally, the dialyzedsamplewas lyophili zed.
2.8 Enrichment of Tropomyosin 2.8. / Ion-ExchangeChromatography
Approximately 300 mg of protein was dissolved in 40 ml of filtered starting buffer (75 mM NaC!, 30 mM Tris, I mM OTI, pH8.0, -10DC). A 2.5 x14 em Q Sepharose Fast Flow column(volumeof-70 ml) (AmerishamBiosciences) was equilibrated with -300 ml ofthe abovebuffer (filtered)at a flow rate of -15ml/hr,in the cold room.The pre- spun proteinsolutionwasloaded onto thecolumnat a rate of - 20 ml/hr. The column was thenwashedwith-100mlof startingbuffer, at the samerate.Theproteinwas eluted fromthecolumnby a saltgradientof 75-500 mM NaC!.Elutionoccurredat a rate of- 30 ml/hrandfractionswerecollectedevery9min.Thecollecte dfractionswere analyzed viaabso rbanceat280 nmandconductivity measurement s (COM80 conductiv ity meter
from Radiom eter)to determinewhich fractionscontained tropomy osin .Fractions believedto containtropomyosinwereconfirmed via SOS 12%PAGE.
2.8.2 Hydr oxy apatite Chromatography
A 2.5x16 cm hydrox yapatite column(volume of -80 ml) (BioRad) was equilibratedat roomtemperaturewith300 ml of filtered starting buffer(IMNaCI,0.0 I%
NaAzide ,30 mMsodium phosph ate,I mMorr,pH 7.0)at a rateof-15ml/hr.The combined tropomyosincontaining fractionsfrom theFastQcolumn weredirectly load ed onto thecolumnat 15mllh r. Thecolumnwas thenwashedwith -100ml of startbutTer.A sodium phosphategradientof 30 mM to 250 mM wasusedto elute theproteinat a rate of - 30 mllhr. Frac tions werecollectedevery9 min.SOS 12%PAGEand absorb ance measurement sat280 nm wereusedtodetermin ethetropom yosin containing fractions.
Thesefraction swerecombinedand dialyzed (w ide dialysistubing,6,000-8,000 Da MWCO)(in thecold room) overtwodaysagain stdH20(containingammo nium bicarbo nateand mercaptoethanol) whilechanging the water four times(foratotalof 20 L). The samplewasthen lyophilized.Theenrichment of the protein was assessedby SOS 12%PAGE.
2.9Proteolyticdigestion
2.9.1 Omp- TDigestion
Outermembraneprotease-T (Omp-T)digestionof tropomyosinwasperformedas describedby Goonasekaraet al., (2007). Approxim ately13 mg of protein was dissolv ed inIml of buffer (0. 1 M NaCI,50 mMsodium phosphat e,5 mMEOTA, I mMorr,pH
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