Supplementary material

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Fig S1: Purification protocol for TBCB, TBCB3, and TBCB9 polypeptides. (a) Schematic diagram showing the different steps followed for the purification of TBCB and the two deletion derivatives of TBCB without the last three or nine residues (TBCB3 and TBCB9). Fractions collected from a Superdex-75 10/300 GL column (equilibrated with buffer B) were analyzed by SDS-PAGE. Lane 1 shows molecular mass markers. Buffer A: 50 mM Mes (pH 6.7), 1 mM DTT, 1 mM MgCl2, 5 mM NaCl, and 0.5 mM PMSF. Buffer B: 50 mM Mes (pH 6.7), 1 mM DTT, 1 mM MgCl2, 5 mM NaCl, and 0.5 mM PMSF.(b) TBCB3 does not depolymerize microtubules in vitro. We studied the interaction between TBCB and preformed microtubules by means of conventional microtubule binding experiments with recombinant TBCB3. Aliquots of pellets containing microtubules and supernatants containing soluble tubulin were analyzed by SDS-PAGE. Lane 1, molecular mass markers. Lanes 2 and 3, supernatant (SN) and pellet (P) in the presence of TBCB3. Lane 5 and 6, supernatant (SN) and pellet (P) of microtubules, and lanes 8 and 9 supernatant (SN) and pellet (P) in the presence of ovalbumin as a negative control.

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Fig S2: TBCE and TBCB, the tubulin heterodimer dissociation machine.

a) Nonclassical two-dimensional native SDS-PAGE of complexes formed in tubulin dissociation experiments by TBCE and TBCB. Aliquots of TBCB (a) or TBCB3 (b) and TBCE and tubulin heterodimers were incubated at 30°C for 30 min and loaded onto a 6% native minigel (1D). After electrophoresis, a single running lane containing the native electrophoresed sample was excised and loaded onto a preparative SDS-minigel (2D, see Materials and Methods). TC, Ternary complex containing TBCE, TBCB and -tubulin. To confirm the composition of this band (panel a, second dimension), we have analyzed the sample where TBCE was incubated with TBCB and -tubulin using a nonclassical two-dimensional PAGE method [23]. Thus the incubation sample was analyzed in a native gel for the first dimension. Then, to resolve the complex’s composition, the respective lane of the native gel was excised and directly applied into a denaturing gel for the second dimension. This analysis confirmed that the new extra band corresponds to the ternary complex. b) Nonclassical two-dimensional native SDS-PAGE of complexes formed in tubulin dissociation experiments by TBCE and TBCB3. C, control of tubulin heterodimers; C1, TBCB3; C2, tubulin dissociation in the presence of TBCE and TBCB3 for 30 min. In this experiment, bands containing this first dimension (1D) were excised from the gel and loaded onto an 8.5% SDS-polyacrylamide gel as described in Materials and Methods (2D). After electrophoresis, the gels were stained with Coomassie Blue. The second dimension (2D) shows the molecular composition of the new band that migrates a bit slower than tubulin heterodimers and corresponds to a binary complex formed between TBCB3 and -tubulin. c) TBCB and TBCB3 do not dissociate the tubulin heterodimer. We decided to repeat the dissociation experiments by performing them in the presence of TBCA that is able to accept β-tubulin upon tubulin heterodimer dissociation and therefore to further demonstrate the occurrence of this dissociation. In fact, TBCA captures the -tubulin subunits emerging from tubulin dimers dissociated by TBCE and forms a binary complex with -tubulin that is detectable in native gels [8]. Aliquots containing TBCB or TBCB3 were incubated at 30°C for 30 min with tubulin heterodimers in the presence and absence of TBCE and in the presence of TBCA. The drawing on the right shows the migration of the different proteins and complexes [5, 8-9]. As observed in panel c, when tubulin heterodimers are incubated with TBCE in the presence of TBCB and TBCA, dissociation is complete. The result is the formation of the ternary complex (TBCB, TBCE, and -tubulin) described above, which comigrates with free TBCA and the binary TBCA/-tubulin complex. On the other hand, when TBCE and tubulin heterodimers are incubated with TBCB3 instead of TBCB, the ternary complex is not detected, but binary complexes containing TBCB3 and -tubulin, and TBCA and -tubulin are clearly visible. In contrast, neither TBCB nor TBCB3 by itself gives rise to the formation of TBCA and -tubulin complexes, demonstrating that they do not have the ability to dissociate the tubulin heterodimer in the absence of TBCE (panel c).d) TBCE slowly dissociates the tubulin heterodimer. Time-course of tubulin dissociation in the presence of stoichiometric amounts of TBCE analyzed by non-denaturing electrophoresis. C, control of tubulin heterodimers incubated for 30 min. Samples were frozen in dry ice until they were loaded into the native gel. e) Time-course of tubulin dissociation in the presence of TBCE and stoichiometric amounts of TBCB or TBCB3 analyzed by non-denaturing electrophoresis. C1, TBCB; C2, TBCB3; C3, control of tubulin heterodimers; C4, tubulin dissociation in the presence of TBCE for 30 min. After the indicated times, samples were frozen on dry ice until

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they were loaded into the native gel. The drawing on the right shows the migration of the different proteins and complexes [5, 8-9]. Final concentrations used were 3 M for tubulin, 2.5 M for TBCE, and 2.5 M for TBCB and TBCB3. f) Quantification of tubulin dissociation by TBCE/TBCB and TBCE/TBCB3. Quantification of the percentage of tubulin heterodimers remaining in the experiment shown in d (dissociation by TBCE) and e (disociation by TBCE/TBCB and by TBCE/TBCB 3) at time 0. The gel was stained with Coomassie blue and scanned before drying. The bands were quantified using ImageJ 1.36b (by Wayne Rasband, NIH, MD, USA) software freely available on the internet. The bar chart was made using SigmaPlot 8.0 software (Systat Software, Richmond, CA,USA). Experiments were performed in triplicate, and the graph reports the mean ± the standard deviation. The composition of the different complexes was always confirmed by western blots using specific antibodies (data not shown, [9]).

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Figure S3

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Fig S3: a) CD spectra (left) and thermal unfolding (right) of TBCB, TBCB3, and TBCB9. All tests were performed at a concentration between 6 M and 10 M. The CD spectra (left) were recorded at 4°C and 30°C. On the right is shown the thermal unfolding as followed by measuring the ellipticity at 220 nm between 4°C and 80°C. b) Analysis of TBCB, TBCB3 and TBCB9 by dynamic light scattering (DLS). The table shows the data obtained by DLS for the hydrodynamic radius (R), percentage of polydispersion (% PD) and the estimated molecular mass (MW-R) for each sample. The final concentrations of the proteins were 55 M of TBCB, 45 M of TBCB3, and 40 M of TBCB9. (c) Schematic diagram showing the different steps followed in the purification of untagged EB1. Diagram showing the different steps in the purification of untagged EB1. The purified protein was analyzed by SDS-PAGE using fractions eluted from the Superdex-75 10/300 GL column equilibrated with buffer B. EB1 eluted as a dimer (elution volume: 8.5 ml). Lane 1 shows molecular weight markers. Buffer A: 20 mM Tris (pH 8.0), 1 mM EDTA, and 0.5 mM PMSF. Buffer B: Bis-Tris 20 mM (pH 7.0), KCl 100 mM, DTT 1 mM, and PMSF 0.5 mM. (d) TBCB3 does not interact directly with EB1. Plots of A280 absorbance against elution volume from the size-exclusion chromatography experiments. The elution profiles of EB1, TBCB3, and the combination of these proteins were analyzed by gel filtration through a Superdex 200 PC 3.2/30 column. Three traces are shown: TBCB3 alone (red), EB1 alone (green), and the combination of TBCB3 and EB1 (blue). The final concentration for both proteins was 13 M.

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Figure S4

Fig S4: Analysis of quaternary structures of TBCB and TBCB 3 by cross-linking with different glutaraldehyde concentrations. Purified proteins at the indicated concentrations were cross-linked and separated by SDS-12% PAGE. Equal amounts of protein were loaded in each lane. Control lanes with proteins with no treatment are indicated. Cross-linked of EB1 at 1.5 M as a control is indicated in the figure.

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Figure S5

a

Microtubule-associated protein RP/EB family member 1 OS=Homo sapiens.Nominal mass (Mr): 30151; Calculated pI value: 5.02Sequence Coverage: 83%

1 MAVNVYSTSV TSDNLSRHDM LAWINESLQL NLTKIEQLCS GAAYCQFMDM 51 LFPGSIALKK VKFQAKLEHE YIQNFKILQA GFKRMGVDKI IPVDKLVKGK 101 FQDNFEFVQW FKKFFDANYD GKDYDPVAAR QGQETAVAPS LVAPALNKPK 151 KPLTSSSAAP QRPISTQRTA AAPKAGPGVV RKNPGVGNGD DEAAELMQQV 201 NVLKLTVEDL EKERDFYFGK LRNIELICQE NEGENDPVLQ RIVDILYATD 251 EGFVIPDEGG PQEEQEEY

ba 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Ac_Ala Val Asn Val Tyr Ser Thr Ser Val Thr Ser Asp Asn Leu Ser Arg y'' 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

Fig S5: (a) Mass spectrometric analysis of band 1 (Fig 5) indicates, with a sequence coverage of 83%, that it corresponds to the plus-end-tracking protein EB1. (b) Annotation of the Ac_Ala-Val-Asn-Val-Tyr-Ser-Thr-Ser-Val-Thr-Ser-Asp-Asn-Leu-

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Ser-Arg peptide of MARE1_HUMAN (Q15691) protein, indicating the discovery of N-terminal acetylation of EB1 in human cells. Mass spectrometric characterization of the N-terminus of HEK-purified EB1 reveals an acetylation modification, with removal of the initiator methionine residue and acetylation of the following alanine. This cotranslational acetylation only occurs in eukaryotes. The acetylation of the N-terminal alanine of proteins is a biological process catalyzed by peptide -N-acetyltransferase or other enzymes of this class, such as ribosomal-protein-alanine N-acetyltransferase (Driessen, 1985). The [M+2]+2 ion with m/z 877.9340 was identified by MASCOT (Matrixscience) as Ac_Ala-Val-Asn-Val-Tyr-Ser-Thr-Ser-Val-Thr-Ser-Asp-Asn-Leu-Ser-Arg peptide with an ion score of 88 (ion score > 34 indicates identity). The spectrum was processed with MaxEntIII software (MassLynx 4.1, Waters) and manually annotated with Peptide/Protein Editor (MassLynx 4.1, Waters). Ammonium ions (capital letters) and y (y), b, and an ion series have been annotated. *Indicates loss of ammonia and ~ loss of water. Ac_ indicates N-terminal acetylation. All annotated ions are indicated in red in the sequence, but some of them are not visible in the spectrum.

Figure S6aTCPB_HUMAN T-complex protein 1 subunit beta OS=Homo sapiens GN=CCT2 PE=1 SV=4Nominal mass (Mr): 57794; Calculated pI value: 6.01

Sequence Coverage: 76%

1 MASLSLAPVN IFKAGADEER AETARLTSFI GAIAIGDLVK STLGPKGMDK 51 ILLSSGRDAS LMVTNDGATI LKNIGVDNPA AKVLVDMSRV QDDEVGDGTT 101 SVTVLAAELL REAESLIAKK IHPQTIIAGW REATKAAREA LLSSAVDHGS

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151 DEVKFRQDLM NIAGTTLSSK LLTHHKDHFT KLAVEAVLRL KGSGNLEAIH 201 IIKKLGGSLA DSYLDEGFLL DKKIGVNQPK RIENAKILIA NTGMDTDKIK 251 IFGSRVRVDS TAKVAEIEHA EKEKMKEKVE RILKHGINCF INRQLIYNYP 301 EQLFGAAGVM AIEHADFAGV ERLALVTGGE IASTFDHPEL VKLGSCKLIE 351 EVMIGEDKLI HFSGVALGEA CTIVLRGATQ QILDEAERSL HDALCVLAQT 401 VKDSRTVYGG GCSEMLMAHA VTQLANRTPG KEAVAMESYA KALRMLPTII 451 ADNAGYDSAD LVAQLRAAHS EGNTTAGLDM REGTIGDMAI LGITESFQVK 501 RQVLLSAAEA AEVILRVDNI IKAAPRKRVP DHHPC

TCPH_HUMANT-complex protein 1 subunit eta OS=Homo sapiens Nominal mass (Mr): 59842; Calculated pI value: 7.55Sequence Coverage: 76%

1 MMPTPVILLK EGTDSSQGIP QLVSNISACQ VIAEAVRTTL GPRGMDKLIV 51 DGRGKATISN DGATILKLLD VVHPAAKTLV DIAKSQDAEV GDGTTSVTLL 101 AAEFLKQVKP YVEEGLHPQI IIRAFRTATQ LAVNKIKEIA VTVKKADKVE 151 QRKLLEKCAM TALSSKLISQ QKAFFAKMVV DAVMMLDDLL QLKMIGIKKV 201 QGGALEDSQL VAGVAFKKTF SYAGFEMQPK KYHNPKIALL NVELELKAEK 251 DNAEIRVHTV EDYQAIVDAE WNILYDKLEK IHHSGAKVVL SKLPIGDVAT 301 QYFADRDMFC AGRVPEEDLK RTMMACGGSI QTSVNALSAD VLGRCQVFEE 351 TQIGGERYNF FTGCPKAKTC TFILRGGAEQ FMEETERSLH DAIMIVRRAI 401 KNDSVVAGGG AIEMELSKYL RDYSRTIPGK QQLLIGAYAK ALEIIPRQLC 451 DNAGFDATNI LNKLRARHAQ GGTWYGVDIN NEDIADNFEA FVWEPAMVRI 501 NALTAASEAA CLIVSVDETI KNPRSTVDAP TAAGRGRGRG RPH

TCPQ_HUMAN Nominal mass (Mr): 60153; Calculated pI value: 5.42Sequence Coverage: 72%

1 MALHVPKAPG FAQMLKEGAK HFSGLEEAVY RNIQACKELA QTTRTAYGPN 51 GMNKMVINHL EKLFVTNDAA TILRELEVQH PAAKMIVMAS HMQEQEVGDG 101 TNFVLVFAGA LLELAEELLR IGLSVSEVIE GYEIACRKAH EILPNLVCCS 151 AKNLRDIDEV SSLLRTSIMS KQYGNEVFLA KLIAQACVSI FPDSGHFNVD 201 NIRVCKILGS GISSSSVLHG MVFKKETEGD VTSVKDAKIA VYSCPFDGMI 251 TETKGTVLIK TAEELMNFSK GEENLMDAQV KAIADTGANV VVTGGKVADM 301 ALHYANKYNI MLVRLNSKWD LRRLCKTVGA TALPRLTPPV LEEMGHCDSV 351 YLSEVGDTQV VVFKHEKEDG AISTIVLRGS TDNLMDDIER AVDDGVNTFK 401 VLTRDKRLVP GGGATEIELA KQITSYGETC PGLEQYAIKK FAEAFEAIPR 451 ALAENSGVKA NEVISKLYAV HQEGNKNVGL DIEAEVPAVK DMLEAGILDT 501 YLGKYWAIKL ATNAAVTVLR VDQIIMAKPA GGPKPPSGKK DWDDDQND

TCPD_HUMAN T-complex protein 1 subunit delta OS=Homo sapiens Nominal mass (Mr): 58401; Calculated pI value: 7.96Sequence Coverage: 68%

1 MPENVAPRSG ATAGAAGGRG KGAYQDRDKP AQIRFSNISA AKAVADAIRT 51 SLGPKGMDKM IQDGKGDVTI TNDGATILKQ MQVLHPAARM LVELSKAQDI 101 EAGDGTTSVV IIAGSLLDSC TKLLQKGIHP TIISESFQKA LEKGIEILTD 151 MSRPVELSDR ETLLNSATTS LNSKVVSQYS SLLSPMSVNA VMKVIDPATA 201 TSVDLRDIKI VKKLGGTIDD CELVEGLVLT QKVSNSGITR VEKAKIGLIQ 251 FCLSAPKTDM DNQIVVSDYA QMDRVLREER AYILNLVKQI KKTGCNVLLI 301 QKSILRDALS DLALHFLNKM KIMVIKDIER EDIEFICKTI GTKPVAHIDQ 351 FTADMLGSAE LAEEVNLNGS GKLLKITGCA SPGKTVTIVV RGSNKLVIEE 401 AERSIHDALC VIRCLVKKRA LIAGGGAPEI ELALRLTEYS RTLSGMESYC

451 VRAFADAMEV IPSTLAENAG LNPISTVTEL RNRHAQGEKT AGINVRKGGI 501 SNILEELVVQ PLLVSVSALT LATETVRSIL KIDDVVNTR

TCPG_HUMAN T-complex protein 1 subunit gamma OS=Homo sapiens Nominal mass (Mr): 61066; Calculated pI value: 6.10Sequence Coverage: 67%

1 MMGHRPVLVL SQNTKRESGR KVQSGNINAA KTIADIIRTC LGPKSMMKML 51 LDPMGGIVMT NDGNAILREI QVQHPAAKSM IEISRTQDEE VGDGTTSVII 101 LAGEMLSVAE HFLEQQMHPT VVISAYRKAL DDMISTLKKI SIPVDISDSD 151 MMLNIINSSI TTKAISRWSS LACNIALDAV KMVQFEENGR KEIDIKKYAR 201 VEKIPGGIIE DSCVLRGVMI NKDVTHPRMR RYIKNPRIVL LDSSLEYKKG 251 ESQTDIEITR EEDFTRILQM EEEYIQQLCE DIIQLKPDVV ITEKGISDLA 301 QHYLMRANIT AIRRVRKTDN NRIARACGAR IVSRPEELRE DDVGTGAGLL 351 EIKKIGDEYF TFITDCKDPK ACTILLRGAS KEILSEVERN LQDAMQVCRN 401 VLLDPQLVPG GGASEMAVAH ALTEKSKAMT GVEQWPYRAV AQALEVIPRT 501 VKLQTYKTAV ETAVLLLRID DIVSGHKKKG DDQSRQGGAP DAGQE

TCPA_HUMAN T-complex protein 1 subunit alpha OS=Homo sapiens Nominal mass (Mr): 60819; Calculated pI value: 5.80Sequence Coverage: 68%

1 MEGPLSVFGD RSTGETIRSQ NVMAAASIAN IVKSSLGPVG LDKMLVDDIG 51 DVTITNDGAT ILKLLEVEHP AAKVLCELAD LQDKEVGDGT TSVVIIAAEL 101 LKNADELVKQ KIHPTSVISG YRLACKEAVR YINENLIVNT DELGRDCLIN 151 AAKTSMSSKI IGINGDFFAN MVVDAVLAIK YTDIRGQPRY PVNSVNILKA 201 HGRSQMESML ISGYALNCVV GSQGMPKRIV NAKIACLDFS LQKTKMKLGV 251 QVVITDPEKL DQIRQRESDI TKERIQKILA TGANVILTTG GIDDMCLKYF 301 VEAGAMAVRR VLKRDLKRIA KASGATILST LANLEGEETF EAAMLGQAEE 351 VVQERICDDE LILIKNTKAR TSASIILRGA NDFMCDEMER SLHDALCVVK 401 RVLESKSVVP GGGAVEAALS IYLENYATSM GSREQLAIAE FARSLLVIPN 451 TLAVNAAQDS TDLVAKLRAF HNEAQVNPER KNLKWIGLDL SNGKPRDNKQ 501 AGVFEPTIVK VKSLKFATEA AITILRIDDL IKLHPESKDD KHGSYEDAVH 551 SGALND

TCPE_HUMAN T-complex protein 1 subunit epsilon OS=Homo sapiens Nominal mass (Mr): 60089; Calculated pI value: 5.45Sequence Coverage: 60%

1 MASMGTLAFD EYGRPFLIIK DQDRKSRLMG LEALKSHIMA AKAVANTMRT 51 SLGPNGLDKM MVDKDGDVTV TNDGATILSM MDVDHQIAKL MVELSKSQDD 101 EIGDGTTGVV VLAGALLEEA EQLLDRGIHP IRIADGYEQA ARVAIEHLDK 151 ISDSVLVDIK DTEPLIQTAK TTLGSKVVNS CHRQMAEIAV NAVLTVADME 201 RRDVDFELIK VEGKVGGRLE DTKLIKGVIV DKDFSHPQMP KKVEDAKIAI 251 LTCPFEPPKP KTKHKLDVTS VEDYKALQKY EKEKFEEMIQ QIKETGANLA 301 ICQWGFDDEA NHLLLQNNLP AVRWVGGPEI ELIAIATGGR IVPRFSELTA 351 EKLGFAGLVQ EISFGTTKDK MLVIEQCKNS RAVTIFIRGG NKMIIEEAKR 401 SLHDALCVIR NLIRDNRVVY GGGAAEISCA LAVSQEADKC PTLEQYAMRA

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451 FADALEVIPM ALSENSGMNP IQTMTEVRAR QVKEMNPALG IDCLHKGTND 501 MKQQHVIETL IGKKQQISLA TQMVRMILKI DDIRKPGESE E

TCPZ_HUMAN T-complex protein 1 subunit zeta OS=Homo sapiens Nominal mass (Mr): 58444; Calculated pI value: 6.23Sequence Coverage: 45%

1 MAAVKTLNPK AEVARAQAAL AVNISAARGL QDVLRTNLGP KGTMKMLVSG 51 AGDIKLTKDG NVLLHEMQIQ HPTASLIAKV ATAQDDITGD GTTSNVLIIG 101 ELLKQADLYI SEGLHPRIIT EGFEAAKEKA LQFLEEVKVS REMDRETLID 151 VARTSLRTKV HAELADVLTE AVVDSILAIK KQDEPIDLFM IEIMEMKHKS 201 ETDTSLIRGL VLDHGARHPD MKKRVEDAYI LTCNVSLEYE KTEVNSGFFY 251 KSAEEREKLV KAERKFIEDR VKKIIELKRK VCGDSDKGFV VINQKGIDPF 301 SLDALSKEGI VALRRAKRRN MERLTLACGG VALNSFDDLS PDCLGHAGLV 351 YEYTLGEEKF TFIEKCNNPR SVTLLIKGPN KHTLTQIKDA VRDGLRAVKN 401 AIDDGCVVPG AGAVEVAMAE ALIKHKPSVK GRAQLGVQAF ADALLIIPKV 451 LAQNSGFDLQ ETLVKIQAEH SESGQLVGVD LNTGEPMVAA EVGVWDNYCV 501 KKQLLHSCTV IATNILLVDE IMRAGMSSLK G

TCPW_HUMAN T-complex protein 1 subunit zeta-2 OS=Homo sapiens Nominal mass (Mr): 58299; Calculated pI value: 6.63Sequence Coverage: 6%

1 MAAIKAVNSK AEVARAQAAL AVNICAARGL QDVLRTNLGP KGTMKMLASG 51 AGDIKLTKDG NVLLDEMQIQ HPTASLIAKV ATAQDDVTGD GTTSNVLIIG 101 ELLKQADLYI SEGLHPRIIA EGFEAAKIKA LEVLEEVKVT KEMKRKILLD 151 VARTSLQTKV HAELADVLTE VVVDSVLAVR RPGYPIDLFM VEIMEMKHKL 201 GTDTKLIQGL VLDHGARHPD MKKRVEDAFI LICNVSLEYE KTEVNSGFFY 251 KTAEEKEKLV KAERKFIEDR VQKIIDLKDK VCAQSNKGFV VINQKGIDPF 301 SLDSLAKHGI VALRRAKRRN MERLSLACGG MAVNSFEDLT VDCLGHAGLV 351 YEYTLGEEKF TFIEECVNPC SVTLLVKGPN KHTLTQVKDA IRDGLRAIKN 401 AIEDGCMVPG AGAIEVAMAE ALVTYKNSIK GRARLGVQAF ADALLIIPKV 451 LAQNAGYDPQ ETLVKVQAEH VESKQLVGVD LNTGEPMVAA DAGVWDNYCV 501 KKQLLHSCTV IATNILLVDE IMRAGMSSLK

Figure S6bTBA1A_HUMAN Tubulin alpha-1A chain OS=Homo sapiens Nominal mass (Mr): 50788; Calculated pI value: 4.94Sequence Coverage: 50%

1 MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN 51 TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHPE QLITGKEDAA

101 NNYARGHYTI GKEIIDLVLD RIRKLADQCT GLQGFLVFHS FGGGTGSGFT 151 SLLMERLSVD YGKKSKLEFS IYPAPQVSTA VVEPYNSILT THTTLEHSDC 201 AFMVDNEAIY DICRRNLDIE RPTYTNLNRL IGQIVSSITA SLRFDGALNV 251 DLTEFQTNLV PYPRIHFPLA TYAPVISAEK AYHEQLSVAE ITNACFEPAN 301 QMVKCDPRHG KYMACCLLYR GDVVPKDVNA AIATIKTKRT IQFVDWCPTG 351 FKVGINYQPP TVVPGGDLAK VQRAVCMLSN TTAIAEAWAR LDHKFDLMYA 401 KRAFVHWYVG EGMEEGEFSE AREDMAALEK DYEEVGVDSV EGEGEEEGEE 451 Y

TBA1B_HUMAN Tubulin alpha-1B chain OS=Homo sapiens Nominal mass (Mr): 50804; Calculated pI value: 4.94Sequence Coverage: 50%

1 MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN 51 TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHPE QLITGKEDAA 101 NNYARGHYTI GKEIIDLVLD RIRKLADQCT GLQGFLVFHS FGGGTGSGFT 151 SLLMERLSVD YGKKSKLEFS IYPAPQVSTA VVEPYNSILT THTTLEHSDC 201 AFMVDNEAIY DICRRNLDIE RPTYTNLNRL ISQIVSSITA SLRFDGALNV 251 DLTEFQTNLV PYPRIHFPLA TYAPVISAEK AYHEQLSVAE ITNACFEPAN 301 QMVKCDPRHG KYMACCLLYR GDVVPKDVNA AIATIKTKRS IQFVDWCPTG 351 FKVGINYQPP TVVPGGDLAK VQRAVCMLSN TTAIAEAWAR LDHKFDLMYA 401 KRAFVHWYVG EGMEEGEFSE AREDMAALEK DYEEVGVDSV EGEGEEEGEE 451 Y

TBA1C_HUMAN Tubulin alpha-1C chain OS=Homo sapiens Nominal mass (Mr): 50548; Calculated pI value: 4.96Sequence Coverage: 47%

1 MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN 51 TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHPE QLITGKEDAA 101 NNYARGHYTI GKEIIDLVLD RIRKLADQCT GLQGFLVFHS FGGGTGSGFT 151 SLLMERLSVD YGKKSKLEFS IYPAPQVSTA VVEPYNSILT THTTLEHSDC 201 AFMVDNEAIY DICRRNLDIE RPTYTNLNRL ISQIVSSITA SLRFDGALNV 251 DLTEFQTNLV PYPRIHFPLA TYAPVISAEK AYHEQLTVAE ITNACFEPAN 301 QMVKCDPRHG KYMACCLLYR GDVVPKDVNA AIATIKTKRT IQFVDWCPTG 351 FKVGINYQPP TVVPGGDLAK VQRAVCMLSN TTAVAEAWAR LDHKFDLMYA 401 KRAFVHWYVG EGMEEGEFSE AREDMAALEK DYEEVGADSA DGEDEGEEY

TBA3E_HUMAN Tubulin alpha-3E chain OS=Homo sapiens Nominal mass (Mr): 50568; Calculated pI value: 5.00Sequence Coverage: 37%

1 MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN 51 TFFSETGAGK HVPRAVFVDL EPTVVDEVRT GTYRQLFHPE QLITGKEDAA 101 SNYARGHYTI GKEIVDLVLD RIRKLADLCT GLQGFLIFHS FGGGTGSGFA 151 SLLMERLSVD YSKKSKLEFA IYPAPQVSTA VVEPYNSILT THTTLEHSDC 201 AFMVDNEAIY DICRRNLDIE RPTYTNLNRL IGQIVSSITA SLRFDGALNV 251 DLTEFQTNLV PYPRIHFPLA TYAPVISAEK AYHEQLSVAE ITNACFEPAN 301 QMVKCDPRHG KYMACCMLYR GDVVPKDVNA AIATIKTKRT IQFVDWCPTG 351 FKVGINYQPP TVVPGGDLAK VQRAVCMLSN TTAIAEAWAR LVHKFDLMYA 401 KWAFVHWYVG EGMEEGEFSE AREDLAALEK DCEEVGVDSV EAEAEEGEAY

TBA8_HUMAN Tubulin alpha-8 chain OS=Homo sapiens Nominal mass (Mr): 50746; Calculated pI value: 4.94Sequence Coverage: 34%

1 MRECISVHVG QAGVQIGNAC WELFCLEHGI QADGTFDAQA SKINDDDSFT 51 TFFSETGNGK HVPRAVMIDL EPTVVDEVRA GTYRQLFHPE QLITGKEDAA

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101 NNYARGHYTV GKESIDLVLD RIRKLTDACS GLQGFLIFHS FGGGTGSGFT 151 SLLMERLSLD YGKKSKLEFA IYPAPQVSTA VVEPYNSILT THTTLEHSDC 201 AFMVDNEAIY DICRRNLDIE RPTYTNLNRL ISQIVSSITA SLRFDGALNV 251 DLTEFQTNLV PYPRIHFPLV TYAPIISAEK AYHEQLSVAE ITSSCFEPNS 301 QMVKCDPRHG KYMACCMLYR GDVVPKDVNV AIAAIKTKRT IQFVDWCPTG 351 FKVGINYQPP TVVPGGDLAK VQRAVCMLSN TTAIAEAWAR LDHKFDLMYA 401 KRAFVHWYVG EGMEEGEFSE AREDLAALEK DYEEVGTDSF EEENEGEEF

TBA4B_HUMANPutative tubulin-like protein alpha-4B OS=Homo sapiens Nominal mass (Mr): 27819; Calculated pI value: 7.71Sequence Coverage: 10%

1 MRHQQTERQD PSQPLSRQHG TYRQIFHPEQ LITGKEDAAN NYAWGHYTIG 51 KEFIDLLLDR IRKLADQCTG LQGFLVFHSL GRGTGSDVTS FLMEWLSVNY 101 GKKSKLGFSI YPAPQVSTAM VQPYNSILTT HTTLEHSDCA FMVDNKAIYD 151 ICHCNLDIER PTYTNLNRLI SQIVSSITAS LRFDGALNVD LTEFQTNLVS 201 YLTSTSPWPP MHQSSLQKRY TTSSCWWQRL PMPALSLPTR W

TBB5_HUMAN Tubulin beta chain OS=Homo sapiens Nominal mass (Mr): 50095; Calculated pI value: 4.78Sequence Coverage: 65%

1 MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLDRISVY 51 YNEATGGKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDNF VFGQSGAGNN 101 WAKGHYTEGA ELVDSVLDVV RKEAESCDCL QGFQLTHSLG GGTGSGMGTL 151 LISKIREEYP DRIMNTFSVV PSPKVSDTVV EPYNATLSVH QLVENTDETY 201 CIDNEALYDI CFRTLKLTTP TYGDLNHLVS ATMSGVTTCL RFPGQLNADL 251 RKLAVNMVPF PRLHFFMPGF APLTSRGSQQ YRALTVPELT QQVFDAKNMM 301 AACDPRHGRY LTVAAVFRGR MSMKEVDEQM LNVQNKNSSY FVEWIPNNVK 351 TAVCDIPPRG LKMAVTFIGN STAIQELFKR ISEQFTAMFR RKAFLHWYTG 401 EGMDEMEFTE AESNMNDLVS EYQQYQDATA EEEEDFGEEA EEEA

TBB2A_HUMAN Tubulin beta-2A chain OS=Homo sapiens Nominal mass (Mr): 50274; Calculated pI value: 4.78Sequence Coverage: 62%

1 MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY 51 YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDNF VFGQSGAGNN 101 WAKGHYTEGA ELVDSVLDVV RKESESCDCL QGFQLTHSLG GGTGSGMGTL 151 LISKIREEYP DRIMNTFSVM PSPKVSDTVV EPYNATLSVH QLVENTDETY 201 SIDNEALYDI CFRTLKLTTP TYGDLNHLVS ATMSGVTTCL RFPGQLNADL 251 RKLAVNMVPF PRLHFFMPGF APLTSRGSQQ YRALTVPELT QQMFDSKNMM 301 AACDPRHGRY LTVAAIFRGR MSMKEVDEQM LNVQNKNSSY FVEWIPNNVK 351 TAVCDIPPRG LKMSATFIGN STAIQELFKR ISEQFTAMFR RKAFLHWYTG 401 EGMDEMEFTE AESNMNDLVS EYQQYQDATA DEQGEFEEEE GEDEA

TBB2B_HUMAN Tubulin beta-2B chain OS=Homo sapiens Nominal mass (Mr): 50377; Calculated pI value: 4.78Sequence Coverage: 62%

1 MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY 51 YNEATGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDNF VFGQSGAGNN 101 WAKGHYTEGA ELVDSVLDVV RKESESCDCL QGFQLTHSLG GGTGSGMGTL

151 LISKIREEYP DRIMNTFSVM PSPKVSDTVV EPYNATLSVH QLVENTDETY 201 CIDNEALYDI CFRTLKLTTP TYGDLNHLVS ATMSGVTTCL RFPGQLNADL 251 RKLAVNMVPF PRLHFFMPGF APLTSRGSQQ YRALTVPELT QQMFDSKNMM 301 AACDPRHGRY LTVAAIFRGR MSMKEVDEQM LNVQNKNSSY FVEWIPNNVK 351 TAVCDIPPRG LKMSATFIGN STAIQELFKR ISEQFTAMFR RKAFLHWYTG 401 EGMDEMEFTE AESNMNDLVS EYQQYQDATA DEQGEFEEEE GEDEA

TBB2C_HUMANTubulin beta-2C chain OS=Homo sapiens Nominal mass (Mr): 50255; Calculated pI value: 4.79Sequence Coverage: 62%

1 MREIVHLQAG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLERINVY 51 YNEATGGKYV PRAVLVDLEP GTMDSVRSGP FGQIFRPDNF VFGQSGAGNN 101 WAKGHYTEGA ELVDSVLDVV RKEAESCDCL QGFQLTHSLG GGTGSGMGTL 151 LISKIREEYP DRIMNTFSVV PSPKVSDTVV EPYNATLSVH QLVENTDETY 201 CIDNEALYDI CFRTLKLTTP TYGDLNHLVS ATMSGVTTCL RFPGQLNADL 251 RKLAVNMVPF PRLHFFMPGF APLTSRGSQQ YRALTVPELT QQMFDAKNMM 301 AACDPRHGRY LTVAAVFRGR MSMKEVDEQM LNVQNKNSSY FVEWIPNNVK 351 TAVCDIPPRG LKMSATFIGN STAIQELFKR ISEQFTAMFR RKAFLHWYTG 401 EGMDEMEFTE AESNMNDLVS EYQQYQDATA EEEGEFEEEA EEEVA

TBB4_HUMAN Tubulin beta-4 chain OS=Homo sapiens Nominal mass (Mr): 50010; Calculated pI value: 4.78Sequence Coverage: 51%

1 MREIVHLQAG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLERINVY 51 YNEATGGNYV PRAVLVDLEP GTMDSVRSGP FGQIFRPDNF VFGQSGAGNN 101 WAKGHYTEGA ELVDAVLDVV RKEAESCDCL QGFQLTHSLG GGTGSGMGTL 151 LISKIREEFP DRIMNTFSVV PSPKVSDTVV EPYNATLSVH QLVENTDETY 201 CIDNEALYDI CFRTLKLTTP TYGDLNHLVS ATMSGVTTCL RFPGQLNADL 251 RKLAVNMVPF PRLHFFMPGF APLTSRGSQQ YRALTVPELT QQMFDAKNMM 301 AACDPRHGRY LTVAAVFRGR MSMKEVDEQM LSVQSKNSSY FVEWIPNNVK 351 TAVCDIPPRG LKMAATFIGN STAIQELFKR ISEQFTAMFR RKAFLHWYTG 401 EGMDEMEFTE AESNMNDLVS EYQQYQDATA EEGEFEEEAE EEVA

TBB3_HUMAN Tubulin beta-3 chain OS=Homo sapiens Nominal mass (Mr): 50856; Calculated pI value: 4.83Sequence Coverage: 38%

1 MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPSGNYVGDS DLQLERISVY 51 YNEASSHKYV PRAILVDLEP GTMDSVRSGA FGHLFRPDNF IFGQSGAGNN 101 WAKGHYTEGA ELVDSVLDVV RKECENCDCL QGFQLTHSLG GGTGSGMGTL 151 LISKVREEYP DRIMNTFSVV PSPKVSDTVV EPYNATLSIH QLVENTDETY 201 CIDNEALYDI CFRTLKLATP TYGDLNHLVS ATMSGVTTSL RFPGQLNADL 251 RKLAVNMVPF PRLHFFMPGF APLTARGSQQ YRALTVPELT QQMFDAKNMM 301 AACDPRHGRY LTVATVFRGR MSMKEVDEQM LAIQSKNSSY FVEWIPNNVK 351 VAVCDIPPRG LKMSSTFIGN STAIQELFKR ISEQFTAMFR RKAFLHWYTG 401 EGMDEMEFTE AESNMNDLVS EYQQYQDATA EEEGEMYEDD EEESEAQGPK

TBB6_HUMAN Tubulin beta-6 chain OS=Homo sapiens Nominal mass (Mr): 50281; Calculated pI value: 4.77Sequence Coverage: 22%

1 MREIVHIQAG QCGNQIGTKF WEVISDEHGI DPAGGYVGDS ALQLERINVY 51 YNESSSQKYV PRAALVDLEP GTMDSVRSGP FGQLFRPDNF IFGQTGAGNN 101 WAKGHYTEGA ELVDAVLDVV RKECEHCDCL QGFQLTHSLG GGTGSGMGTL 151 LISKIREEFP DRIMNTFSVM PSPKVSDTVV EPYNATLSVH QLVENTDETY 201 CIDNEALYDI CFRTLKLTTP TYGDLNHLVS ATMSGVTTSL RFPGQLNADL 251 RKLAVNMVPF PRLHFFMPGF APLTSRGSQQ YRALTVPELT QQMFDARNMM

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301 AACDPRHGRY LTVATVFRGP MSMKEVDEQM LAIQSKNSSY FVEWIPNNVK 351 VAVCDIPPRG LKMASTFIGN STAIQELFKR ISEQFSAMFR RKAFLHWFTG 401 EGMDEMEFTE AESNMNDLVS EYQQYQDATA NDGEEAFEDE EEEIDG

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Fig S6: Mass spectrometric analysis of bands 2, 3, 4, and 5 (Fig 5).Band 2 contains all of the different - and -tubulin isotypes that are expressed in the human cell line. Band 6 contains Hsp90 and subunits - and . Samples from the affinity column were analyzed in 10% SDS gels, and under these conditions - and -tubulins migrate in the same band. We were able to identify five -tubulin isotypes and seven -tubulin isotypes. (5a) UniProtKB/Swiss-Prot sequence accession numbers are as follows: TCPB, P78371; TCPA, P17987; TCPH, Q99832; TCPE, P48643; TCPQ, P50990; TCPZ, P40227; TCPD, P50991; TCPW, Q92526; and TCPG, P49368. (5b) UniProtKB/Swiss-Prot sequence accession numbers are as follows: TBA1A, Q71U36; TBA1B, P68363; TBA1C, Q9BQE3; TBA3E, Q6PEY2; TBA8, Q9NY65; TBA4B, Q9H853; TBB5, P07437; TBB2A, Q13885; TBB2B, Q9BVA1; TBB2C, P68371; TBB4, P04350; TBB3, Q13509; and TBB6, Q9BUF5.

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Figure S7

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Fig S7: (a) The experimental concentrations of the stocks of FL-peptides were determined by UV spectroscopy: FL-peptide 1: 6.0 M; FL-peptide 2: 7.6 M; FL-peptide 3: 3 M; and FL-peptide 4: 10 M. For KD determination, these experimental concentrations were used throughout data analysis.(b) For FP binding assays, the minimal optimal concentration of FL-peptides was determined by measuring the polarization of dilution series of FL-peptides (n = 3). The concentration of 1 M fluorophore ensures a stable polarization signal and was used for FP binding assays. (c, d) Binding assays of TBCB with FL-peptide 3 or 4 were performed with a maximum protein concentration of 100 M. Data from the FP binding assays of TBCB with FL-peptide 1 or 2 were reanalyzed with the proper fluorescence quenching correction factor (Q). KD values were estimated by fixing the maximum binding to rB = 0.13. (*) No saturation.

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Table S1.- Fluorescein labeled peptides for fluorescence polarization (FP) binding

assays.

Sequence Mm (Da)

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fluorescein [fl] 372

FL-Peptide 1 [fl]-[C6 spacer]-EEDYGLDEI 1585

FL-Peptide 2 [fl]-[C6 spacer]-EEDYGL 1227

FL-Peptide 3 [fl]-[C6 spacer]-GEGEEEGEEY 1629

FL-Peptide 4 [fl]-[C6 spacer]-GEGEEEGEE 1466

Supplemental Experimental ProceduresPrediction of the structure of the C-terminal tail (9 residues) of human TBCB.The structure of the last nine residues of human TBCB was predicted with the Pepstr server (http://www.imtech.res.in/raghava/pepstr/home.html). This server predicts the tertiary structure of small peptides with sequence length varying between 7 and 25 residues. The prediction strategy is based on the understanding that the -turn is an important and consistent feature of small peptides in addition to regular structures. Thus, the method uses both the regular secondary structure information predicted from PSIPRED and -turn information predicted from BetaTurns software. The side-chain angles were placed using a standard backbone-dependent rotamer library. The structure was further refined with energy minimization and molecular dynamic simulations using Amber software version 6.

TBCAHis purification.TBCAHis was purified from a 50 mL culture of Sf9 cells infected with baculoviruses carrying the human TBCAHis cDNA cloned. Cells were pelleted by centrifugation, washed, and stored frozen at –70°C. Pellets were resuspended in 7.5 mL of 0.5 mM Tris buffer (pH 8) containing protease inhibitors and sonicated three times during 20 seconds at 4°C. Extract was spun at 60,000 g in a Ti 50.2 ultracentrifuge rotor (Beckman-Coulter) for 30 min at 4°C. Supernatant was supplemented with a buffer stock to produce final concentrations of 50 mM Tris-buffer (pH 8) and 500 mM NaCl and 10 mM imidazole and loaded into a 1 mL His-Trap column (GE Healthcare). Fractions containing TBCA–His were pooled and concentrated by ultrafiltration using an Amicon Ultra 10K filter (Millipore, USA). Concentrated protein was applied to a high-resolution gel-filtration column (Superdex-75 HR, GE Healthcare, USA), equilibrated and eluted with 20 mM Bis-Tris buffer (pH 7) containing 100 mM KCl, 1 mM DTT and 0.5 mM PMSF at 0.4 mL/min.

Mass spectrometric analysisSelected protein bands were manually excised from the gel and subjected to in-gel tryptic digestion according to Shevchenko et al. (1996), with minor modifications. The gel pieces were swollen in digestion buffer containing 50 mM NH4HCO3 and 12.5 ng/L proteomics grade trypsin (Roche, Basel, Switzerland) in an ice bath. After 45 min, the supernatant was discarded, and 5 L of 50 mM NH4HCO3 were added to the gel pieces. Digestion proceeded at 37°C overnight. The supernatant was recovered, and peptides were extracted twice: first, with 25 mM NH4HCO3 and acetonitrile (ACN), and second with 0.1% trifluoroacetic acid (TFA) and ACN. The recovered supernatants and extracted peptides were pooled, dried in a SpeedVac concentrator, redissolved in 10 L of 0.1% formic acid (FA), and sonicated for 5 min.Tandem mass spectra were acquired using a SYNAPT HDMS mass spectrometer (Waters, Milford, MA, USA) interfaced with a nanoAcquity UPLC System (Waters). An aliquot (8 L) of each sample was loaded onto a Symmetry 300 C18 (180 m 20 mm) precolumn (Waters) and washed with 0.1% FA for 3 min at a flow rate of 5 L/min. The precolumn was connected to a BEH130 C18, (75 m 200 mm) column (Waters) equilibrated in 3% ACN and 0.1% FA. Peptides were eluted with a 30 min linear gradient of 3%–60% ACN directly onto a nanoelectrospray capillary tip. The capillary voltage was set to 3500 V, and data-dependent MS/MS acquisitions were performed on precursors with charge states of 2, 3, or 4 over a survey m/z range of 350–1990.

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Obtained spectra were processed using VEMS (Matthiesen et al., 2005) and searched against the SwissProt database (version 2011_03) using Mascot (Matrix Science, London, UK).

Circular dichroism (CD) spectroscopyAll CD measurements were acquired on a Jasco-810 spectropolarimeter equipped with a peltier thermoelectric temperature controller. CD spectra were obtained between 260 and 200 nm, with a 0.2 mm path-length, a response time of 4 s, and a bandwidth of 1 nm. Protein samples were studied at concentrations of between 6 and 10 M in PBS. The spectra reported are the averages of three scans, corrected by subtracting the CD spectra of the buffer.Thermal stability was monitored by measuring the ellipticity at 220 nm between 4 and 80°C, with a heating rate of 1°C min–1. The transition temperature (Tm) was estimated by direct nonlinear least-squares fitting of the temperature dependent ellipticity 220 according to a two-state model (Greenfield, 2006).The thermal stability of TBCB, TBCB3, and TBCB9 was then monitored by measuring the ellipticity at 220 nm between 4°C and 80°C. Although the calculated temperature for unfolding is the same (58°C) for the three proteins, noticeable differences are observed between the three unfolding curves (Fig S2A, right panels). TBCB and TBCB3 show an anomalous loss of ellipticity with increasing temperature above the midpoint of the unfolding transition, whereas the thermal denaturation of TBCB9 shows two transitions. The first transition (from 10°C to about 55°C) parallels those of the other two proteins, with a gain in ellipticity at 220 nm that suggests the dissociation of soluble aggregates or the conversion from a molten globule to a state with a higher (and nonnative) -helical content, or both. In the second transition (55°C to 80°C), a typical complete loss of ellipticity dominates the melting curve. This transition occurs in a narrow range of temperature, which is an indication of a cooperative process. These results can be explained by assuming that the common C-terminal motif (EEDYGL) present in TBCB, which is retained in TBCB3 but is absent in TBCB9, is responsible for the aggregation between unfolded monomers, leading to an increase in ellipticity, and/or for their precipitation, resulting in light scattering that interfere with the CD signal for TBCB and TBCB3.

Dynamic light scatteringDynamic light scattering (DLS) data were collected using a DynaPro instrument at an angle of = 90° and processed using Dynamics software version 6 (Protein Solutions). A regularization algorithm was used to resolve multimodal populations and their polydispersity (%PD), normalized to the mean size of the peak and the hydrodynamic radius (RH). All RH data reported were modeled as (isotropic) spheres. Protein samples at a concentration of 40 M in PBS were centrifuged at 4°C and 16000 g prior to measurement. DLS measurements were performed at 4°C and consisted of 70–100 acquisitions of 10 s each.The analysis of the DLS data shows that TBCB and TBCB3 have a degree of polydispersion close to 20%, indicating a high homogeneity of the samples, whereas this value in TBCB9 is 28%, indicative of a higher degree of size heterogeneity (Fig S2B). Several studies have shown that the addition of Ca2+ to calmodulin (CaM) increased its hydrodynamic radius from 2.5 to 3.0 nm. On the other hand, different peptides studied to date induced a collapse of the elongated Ca2+–CaM structure to a more globular form, by decreasing its hydrodynamic radius by an average of 25% (Papish et al., 2002). By analogy, we propose that the last disorganized residues of

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TBCB contribute to an increase in the hydrodynamic value (from 2.7 to 3.6 nm), which could explain why wild-type TBCB presents, at least in gel filtration and SDS–PAGE analyses, a higher than predicted molecular mass. The unstructured peptide at the C terminus compromises TBCB behavior in dynamic light scattering and in circular dichroism.Fluorescence polarization binding assaysThe different fluorescein labeled (FL)-peptide sequences examined are summarized in Supp Table 1. These FL-peptides were dissolved in binding buffer (20 mM potassium phosphate, 50 mM KCl, 1 mM TCEP), and the concentrations were determined by UV (Fig S5A) exploiting the absorbance properties of coupled fluorescein (492 nm = 73900 M 1 cm–1; Hafner, 2008).The dissociation constants for the FL-peptides were determined from the change in anisotropy of the FL-peptide as the protein–peptide complex is formed. The optimal minimal concentration of FL-peptide that gives a stable polarization signal, avoiding measurement artifact (scattering), was determined empirically by measuring the fluorescence polarization of a serially diluted FL-peptide sample (Hafner, 2008; Fig S5B). Solutions that contained 1 M FL-peptide and various concentrations (0–8 M) of protein samples were prepared in a total of 50 L of binding buffer. Following incubation at 30°C for 30 min, the fluorescence intensities parallel (IVV) and perpendicular (IVH) to the plane of excitation were measured in a black 96-well assay plate (Corning) on a Wallac Victor2V 1420 multilabel counter (PerkinElmer) using a 485 nM excitation filter, a 535 nM emission filter, a 1 s per well reading time, and a Z height set at 4 mm. The steady-state fluorescence anisotropy (r) was calculated using equation 1:

r=IVV−GI VHIVV+2GIVH (1)

where the correction factor G (G = 1.12 determined empirically using a fluorescein standard) was introduced to correct the polarized light transmission efficiency of the instrument optics (Lacowicz, 2006).The fluorescence quantum yield of the FL-peptides did not change upon binding to the protein, and the molar fraction of bound (FSB) FL-peptides was calculated following the principle of additivity of anisotropies:

FSB=r−rFrB−rF (2)

where r is the observed anisotropy, and rF and rB are the anisotropies of the free and bound FL-peptides, respectively (Tetin, 2000). The value of rB = 0.13 (obtained from complete titration curves of other peptides) was used for the determination of KD for the protein-FL-peptide complexes that did not reach the saturation condition. After normalization, the dissociation constant (KD) was determined by data fitting (Prism, GraphPad) to the following equation:

FSB=1

2 LT[( LT+PT+K D )−√( LT+PT+KD )2−(4 LT PT ) ]

(3)where LT is the total FL-peptide concentration and PT is the total protein concentration (Roehrl, 2004).In the particular case of FL-peptide 3 or 4, where concentrations up to 100 M were used, the fraction bound FSB was calculated taking into account the proper fluorescence quenching correction factor Q (Roehrl, 2004):

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FSB=r−rF

(rB−rF )Q+r−rF (4)

Analytical ultracentrifugationSedimentation velocity experiments were performed with a XL-A analytical ultracentrifuge (Beckman-Coulter Inc.). A total of 390 µl samples at a concentration of 150 µM, were used in a standard 12 mm charcoal-filled Epon double-sector centrepiece equipped with sapphire windows, inserted in an An50 Ti eight-hole rotor. Absorbance data were acquired at rotor speed of 42000 rpm and at a temperature of 20°C. Data were modeled as a superposition of Lamm equation solutions with the SEDFIT software (Schuck , 2000). The sedimentation coefficient distribution, c(s), was calculated at a confidence level of p = 0.68. The experimental sedimentation values were determined by integration of the main peak of c(s) and corrected to standard conditions to get the corresponding s20,w values with the SEDNTERP program. Calculation of frictional coefficient ratio was performed with the SEDFIT program to obtain the c(M) distribution (Schuck, 2000).

Supplemental References

Borgstahl, G.E. (2007). How to use dynamic light scattering to improve the likelihood of growing macromolecular crystals. Methods Mol. Biol. 363,109-129.Driessen, H. P., de Jong, W. W., Tesser, G. I., and Bloemendal, H.(1985). The mechanism of N-terminal acetylation of proteins. CRC Crit. Rev. Biochem. 18, 281–325.Greenfield, N.J. (2006a). Analysis of the kinetics of folding of proteins and peptides using circular dichroism. Nat Protoc.1, 2891-2899.Greenfield, N.J. (2006b). Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 1, 2876-2890.Hafner, M., Vianini, E., Albertoni, B., Marchetti, L., Grüne, I., Gloeckner, C., and Famulok, M. (2008). Displacement of protein-bound aptamers with small molecules screened by fluorescence polarization. Nature. Protocols, 3, 579-587.Lacowicz, J.R. (2006). Principles of fluorescence spectroscopy, Springer, 3rd Edition.Matthiesen, R., Sorensen, M., Bunkenborg, J., Hojrup, P. and Jensen, O.N. (2005). VEMS 3.0: Algorithms and computational tools for tandem mass spectrometry based identification of post-translational modifications in proteins. J. Proteome Res. 4, 2338-2347.Prystay, L., Gosselin, M., and Banks, P. L.(2001). Determination of equilibrium dissociation constants in fluorescence polarization, J. Biomol. Screening. 6, 141-150.Roehrl, M.H., Wang, J.W., and Wagner, G. (2004). A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein-protein interactions by fluorescence polarization, Biochemistry, 43, 16056-16066.Segur J.B., and Oberstar, H.E. (1951). Viscosity of glycerol and its aqueous solutions, Ind. Eng. Chem. 43, 2117-2120.

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Shevchenko, A., Wilm, M., Vorm, O. and Mann, M. (1996). Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem., 68, 850–858.Zhang, J.H., Chung, T.D., and Oldenburg K.R. (1999). A simple statistical parameters to use in evaluation and validation of high throughput screening assays. J. Biomol. Screening. 4, 67-73.Schuck, P. (2000). Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modelling. Biophys. J. 78, 1606–1619.

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