IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Dimerization of Toc-GTPases at the chloroplast protein import machinery. Dimerization of Toc-GTPases at the chloroplast protein import machinery. J. Biol. Chem. (0021-9258) 2003 Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch 25-Jun-2008: Contacted by JYOTI. in vitro In vitro bn-page blue native page bn-page western blot western blot Immuno blot Immuno blot Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch Accepted 2008-MAY-25 by JYOTI. 25-Jun-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2003 Dimerization of Toc-GTPases at the chloroplast protein import machinery. Dimerization of Toc-GTPases at the chloroplast protein import machinery. J. Biol. Chem. (0021-9258) 2003 Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch 25-Jun-2008: Contacted by JYOTI. in vitro In vitro gtpase assay gtpase assay GTPase gtp hydrolisis gtp hydrolisis GTPase weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch Accepted 2008-MAY-25 by JYOTI. 25-Jun-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2003 Dimerization of Toc-GTPases at the chloroplast protein import machinery. Dimerization of Toc-GTPases at the chloroplast protein import machinery. J. Biol. Chem. (0021-9258) 2003 Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch 25-Jun-2008: Contacted by JYOTI. in vitro In vitro bn-page blue native page bn-page weight by comassie molecular weight estimation by coomasie staining weight by comassie Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch Accepted 2008-MAY-25 by JYOTI. 25-Jun-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2003 Dimerization of Toc-GTPases at the chloroplast protein import machinery. Dimerization of Toc-GTPases at the chloroplast protein import machinery. J. Biol. Chem. (0021-9258) 2003 Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch 25-Jun-2008: Contacted by JYOTI. in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch Accepted 2008-MAY-25 by JYOTI. 25-Jun-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2003 Dimerization of Toc-GTPases at the chloroplast protein import machinery. Dimerization of Toc-GTPases at the chloroplast protein import machinery. J. Biol. Chem. (0021-9258) 2003 Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch 25-Jun-2008: Contacted by JYOTI. in vitro In vitro bn-page blue native page bn-page weight by staining molecular weight estimation by staining weight by staining Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch Accepted 2008-MAY-25 by JYOTI. 25-Jun-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2003 Dimerization of Toc-GTPases at the chloroplast protein import machinery. Dimerization of Toc-GTPases at the chloroplast protein import machinery. J. Biol. Chem. (0021-9258) 2003 Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch 25-Jun-2008: Contacted by JYOTI. in vitro In vitro filter binding filter binding Filter overlay assay Filter overlay assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch Accepted 2008-MAY-25 by JYOTI. J. Biol. Chem. (0021-9258) 2003 Dimerization of Toc-GTPases at the chloroplast protein import machinery. Dimerization of Toc-GTPases at the chloroplast protein import machinery. J. Biol. Chem. (0021-9258) 2003 Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch 25-Jun-2008: Contacted by JYOTI. in vitro In vitro competition binding competition binding weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Weibel P., Hiltbrunner A., Brand L., Kessler F. In the GTP binding assay, recombinant proteins were incubated with radioactive [{alpha}-32P]GTP. Aliquots of the reactions were applied to nitrocellulose and washed with buffer. Protein together with bound radioactive nucleotides remained attached to the nitrocellulose. felix.kessler@unine.ch Accepted 2008-MAY-25 by JYOTI. 25-Jun-2008: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2003 gdp guanosine 5-diphosphate small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) toc33_arath Translocase of chloroplast 33, chloroplastic TOC33 T7I23.11 At1g02280 PPI1 33 kDa chloroplast outer envelope protein Plastid protein import 1 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MGSLVREWVGFQQFPAATQEKLIEFFGKLKQKDMNSMTVLVLGKGGVGKSSTVNSLIGEQVVRVSPFQAEGLRPVMVSRTMGGFTINIIDTPGLVEAGYVNHQALELIKGFLVNRTIDVLLYVDRLDVYRVDELDKQVVIAITQTFGKEIWCKTLLVLTHAQFSPPDELSYETFSSKRSDSLLKTIRAGSKMRKQEFEDSAIAVVYAENSGRCSKNDKDEKALPNGEAWIPNLVKAITDVATNQRKAIHVDKKMVDGSYSDDKGKKLIPLIIGAQYLIVKMIQGAIRNDIKTSGKPL E48892E123BA412D gtp guanosine triphosphate - GTP 5'-GTP GTP Guanosine 5'-triphosphate guanosine 5'-triphosphoric acid guanosine triphosphate GUANOSINE-5'-TRIPHOSPHATE H4gtp guanosine 5'-(tetrahydrogen triphosphate) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) InChI=1/C10H16N5O14P3/c11-10-13-7-4(8(18)14-10)12-2-15(7)9-6(17)5(16)3(27-9)1-26-31(22,23)29-32(24,25)28-30(19,20)21/h2-3,5-6,9,16-17H,1H2,(H,22,23)(H,24,25)(H2,19,20,21)(H3,11,13,14,18)/t3-,5-,6-,9-/m1/s1/f/h14,19-20,22,24H,11H2 InChI=1/C10H16N5O14P3/c11-10-13-7-4(8(18)14-10)12-2-15(7)9-6(17)5(16)3(27-9)1-26-31(22,23)29-32(24,25)28-30(19,20)21/h2-3,5-6,9,16-17H,1H2,(H,22,23)(H,24,25)(H2,19,20,21)(H3,11,13,14,18)/t3-,5-,6-,9-/m1/s1/f/h14,19-20,22,24H,11H2 tc159_arath Translocase of chloroplast 159, chloroplastic T14P8.24 At4g02510 TOC159 PPI2 TOC160 TOC86 Translocase of chloroplast 160, chloroplastic Translocase of chloroplast 86, chloroplastic 159 kDa chloroplast outer envelope protein Plastid protein import 2 T10P11.19 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MDSKSVTPEPTNPFYASSGQSGKTYASVVAAAAAAAADKEDGGAVSSAKELDSSSEAVSGNSDKVGADDLSDSEKEKPNLVGDGKVSDEVDGSLKEDSTTPEATPKPEVVSGETIGVDDVSSLSPKPEAVSDGVGVVEENKKVKEDVEDIKDDGESKIENGSVDVDVKQASTDGESESKVKDVEEEDVGTKKDDEGESELGGKVDVDDKSDNVIEEEGVELTDKGDVIVNSSPVESVHVDVAKPGVVVVGDAEGSEELKINADAETLEVANKFDQIGDDDSGEFEPVSDKAIEEVEEKFTSESDSIADSSKLESVDTSAVEPEVVAAESGSEPKDVEKANGLEKGMTYAEVIKAASAVADNGTKEEESVLGGIVDDAEEGVKLNNKGDFVVDSSAIEAVNVDVAKPGVVVVGDVEVSEVLETDGNIPDVHNKFDPIGQGEGGEVELESDKATEEGGGKLVSEGDSMVDSSVVDSVDADINVAEPGVVVVGAAKEAVIKEDDKDDEVDKTISNIEEPDDLTAAYDGNFELAVKEISEAAKVEPDEPKVGVEVEELPVSESLKVGSVDAEEDSIPAAESQFEVRKVVEGDSAEEDENKLPVEDIVSSREFSFGGKEVDQEPSGEGVTRVDGSESEEETEEMIFGSSEAAKQFLAELEKASSGIEAHSDEANISNNMSDRIDGQIVTDSDEDVDTEDEGEEKMFDTAALAALLKAATGGGSSEGGNFTITSQDGTKLFSMDRPAGLSSSLRPLKPAAAPRANRSNIFSNSNVTMADETEINLSEEEKQKLEKLQSLRVKFLRLLQRLGHSAEDSIAAQVLYRLALLAGRQAGQLFSLDAAKKKAVESEAEGNEELIFSLNILVLGKAGVGKSATINSILGNQIASIDAFGLSTTSVREISGTVNGVKITFIDTPGLKSAAMDQSTNAKMLSSVKKVMKKCPPDIVLYVDRLDTQTRDLNNLPLLRTITASLGTSIWKNAIVTLTHAASAPPDGPSGTPLSYDVFVAQCSHIVQQSIGQAVGDLRLMNPSLMNPVSLVENHPLCRKNREGVKVLPNGQTWRSQLLLLCYSLKVLSETNSLLRPQEPLDHRKVFGFRVRSPPLPYLLSWLLQSRAHPKLPGDQGGDSVDSDIEIDDVSDSEQEDGEDDEYDQLPPFKPLRKTQLAKLSNEQRKAYFEEYDYRVKLLQKKQWREELKRMKEMKKNGKKLGESEFGYPGEEDDPENGAPAAVPVPLPDMVLPPSFDSDNSAYRYRYLEPTSQLLTRPVLDTHGWDHDCGYDGVNAEHSLALASRFPATATVQVTKDKKEFNIHLDSSVSAKHGENGSTMAGFDIQNVGKQLAYVVRGETKFKNLRKNKTTVGGSVTFLGENIATGVKLEDQIALGKRLVLVGSTGTMRSQGDSAYGANLEVRLREADFPIGQDQSSFGLSLVKWRGDLALGANLQSQVSVGRNSKIALRAGLNNKMSGQITVRTSSSDQLQIALTAILPIAMSIYKSIRPEATNDKYSMY 04AEDE84C1BED3F6 toc33-gtp 6 10 unspecified role unspecified role neutral component neutral component 9 unspecified role unspecified role neutral component neutral component n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction Figure 2 GTP was radiolabelled with alpha P32 At 100 nM [{alpha}-32P]GTP both Toc33-wt and Toc33-R130A bound [{alpha}-32P]GTP with similar efficiency (Fig. 2A). The binding appeared to be specific as a bovine serum albumin control failed to retain [{alpha}-32P]GTP. These results suggest that both Toc33-wt and Toc33-R130A bind GTP with high affinity. The authors conclude that "Toc33-wt and Toc33-R130A bind GTP with high affinity". toc33-gtp-1 7 10 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range O23680:p.Arg130Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain R A Wed May 02 18:28:39 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg130ala' Tue Jun 30 19:35:06 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg130Ala' ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction Figure 2B GTP was radiolabelled with alpha P32 At 100 nM [{alpha}-32P]GTP both Toc33-wt and Toc33-R130A bound [{alpha}-32P]GTP with similar efficiency (Fig. 2A). The binding appeared to be specific as a bovine serum albumin control failed to retain [{alpha}-32P]GTP. These results suggest that both Toc33-wt and Toc33-R130A bind GTP with high affinity. The authors conclude that "the data suggest that GTP-binding to Toc33 may be saturated at cellular concentrations of GTP (>= 0.1 mM) ". GDP GTP Dissociation constant (Kd) 2.6 micromolar wild type Toc33 and Kd 3.8 micromolar Toc33 mutant form Arg130Ala. toc33-gdp 7 8 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range O23680:p.Arg130Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain R A Wed May 02 18:54:23 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg130ala' Tue Jun 30 16:22:02 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg130Ala' ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction Figure 2B GTP was radiolabelled with alpha P32 At 100 nM [{alpha}-32P]GTP both Toc33-wt and Toc33-R130A bound [{alpha}-32P]GTP with similar efficiency (Fig. 2A). The binding appeared to be specific as a bovine serum albumin control failed to retain [{alpha}-32P]GTP. These results suggest that both Toc33-wt and Toc33-R130A bind GTP with high affinity. GDP GTP Dissociation constant (Kd) 4.5 micromolar wild type Toc33 and Kd 8.8 micromolar Toc33 mutant form Arg130Ala. toc33-gtp-2 2 9 unspecified role unspecified role neutral component neutral component n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecobb Escherichia coli (strain B / BL21) 10 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Figure 3 GTP was radiolabelled with alpha P32 The results suggest that both Toc33-wt (Fig. 3A, lanes 1�4) and Toc33-R130A (Fig. 3A, lanes 5�8) hydrolyze GTP, at a low concentration, with similar efficiencies. The authors conclude that "both Toc33-wt (Fig. 3A, lanes 1�4) and Toc33-R130A (Fig. 3A, lanes 5�8) hydrolyze GTP, at a low concentration, with similar efficiencies. ". First-order rate constant, (Kcat) 0.013 per minute micromolar wild type Toc33. toc33-toc33 3 9 unspecified role unspecified role neutral component neutral component n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range O23680:p.Arg130Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain R A Wed May 02 18:41:59 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg130ala' Tue Jun 30 19:10:02 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg130Ala' ecobb Escherichia coli (strain B / BL21) 9 unspecified role unspecified role neutral component neutral component c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain O23680:p.Arg130Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain R A Wed May 02 18:29:53 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg130ala' Tue Jun 30 18:50:45 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg130Ala' ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction Figure 4A In contrast to Toc33-wt, the putative 70-kDa dimer band of Toc33-R130A was much weaker (Fig. 4A, lanes 6-9, dimer), suggesting reduced dimerization when compared with Toc33-wt toc33-toc33-1 1 9 unspecified role unspecified role neutral component neutral component c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain O23680:p.Arg130Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain R A Wed May 02 16:33:05 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg130ala' Tue Jun 30 19:53:30 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg130Ala' ecobb Escherichia coli (strain B / BL21) 9 unspecified role unspecified role neutral component neutral component n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range O23680:p.Arg130Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain R A Wed May 02 17:08:23 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg130ala' Tue Jun 30 17:14:08 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg130Ala' ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction Figure 4B The result suggests that the upper bands also largely consist of Toc33-wt and Toc33-R130A, respectively, most likely as dimers. However, the upper band of Toc33-R130A gave a much weaker signal than that of Toc33-wt, likely due to reduced dimer formation and/or streaking of the protein across the length of the gel (Fig. 4A, lanes 6�9). toc33-toc33-2 5 9 unspecified role unspecified role neutral component neutral component n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range O23680:p.Arg130Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain R A Wed May 02 15:12:21 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg130ala' Tue Jun 30 17:11:54 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg130Ala' ecobb Escherichia coli (strain B / BL21) 9 unspecified role unspecified role neutral component neutral component c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain O23680:p.Arg130Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain R A Wed May 02 20:10:11 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg130ala' Tue Jun 30 17:28:15 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg130Ala' ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction Figure 4B The result suggests that the upper bands also largely consist of Toc33-wt and Toc33-R130A, respectively, most likely as dimers. However, the upper band of Toc33-R130A gave a much weaker signal than that of Toc33-wt, likely due to reduced dimer formation and/or streaking of the protein across the length of the gel (Fig. 4A, lanes 6�9). toc33-toc33-3 4 9 unspecified role unspecified role bait bait c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) 9 unspecified role unspecified role prey prey n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain O23680:p.Arg130Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain R A Wed May 02 16:52:31 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg130ala' Tue Jun 30 19:19:38 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg130Ala' region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction Figure 5 Whereas recombinant Toc33-wt pulled down around 20% of the synthetic, radioactive wild-type protein ([35S]wt) (Fig. 5A, lanes 1�6), recombinant Toc33-R130A pulled down only trace amounts of its radioactive equivalent toc33-toc159 4 11 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 9 unspecified role unspecified role bait bait c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain O23680:p.Arg130Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain R A Wed May 02 18:58:27 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg130ala' Tue Jun 30 17:05:57 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg130Ala' ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction Figure 6 In the pull-down assay, Toc33-wt (Fig. 6A, lanes 1�4) bound more efficiently to soluble [35S]Toc159 (~5% of the total radioactive protein) than Toc33-R130A (~2% of the total radioactive protein) (Fig. 6A, lanes 5�8) suggesting that heterodimerization of Toc33 and Toc159 may also require Arg130 of Toc33 (Fig. 6, A and B). It cannot be inferred from Fig 7 that the mutant form of Toc159 is any better or worse at interacting with wt Toc33 than wt Toc159.