Identification of the photocrosslinking sites in troponin-I with 4-maleimidobenzophenone labelled mutant troponin-Cs having single cysteines at positions 158 and 21
Our previous studies have shown that 4-maleimidobenzophenone (BP-Mal) attached to troponin-C (TnC) mutants with single cysteines at positions 12, 57, 89 and 98 forms crosslinks to troponin-I (TnI), and the identified crosslinking regions indicate an antiparallel course of the two interacting polypep...
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| Veröffentlicht in: | Journal of muscle research and cell motility 1998-06, Vol.19 (5), p.479-490 |
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| container_title | Journal of muscle research and cell motility |
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| creator | Leszyk, J Tao, T Nuwaysir, L M Gergely, J |
| description | Our previous studies have shown that 4-maleimidobenzophenone (BP-Mal) attached to troponin-C (TnC) mutants with single cysteines at positions 12, 57, 89 and 98 forms crosslinks to troponin-I (TnI), and the identified crosslinking regions indicate an antiparallel course of the two interacting polypeptide chains, in agreement with other studies using fragments of TnC and TnI. In this work we extended the mapping of the TnC-TnI interface by analysing photocrosslinking between TnI and BP-Mal labelled TnC mutants with single Cys residues at positions 21 (TnC21) and 158 (TnC158). We determined the sites of these photocrosslinks in TnI by progressive proteolysis of the crosslinked product, followed by N-terminal sequencing and mass spectrophotometric analyses. The results show that whereas TnC158 forms a specific crosslink with Met-21, TnC21 forms multiple crosslinks in the range of residues 96 to 134 of TnI. The results are discussed in light of the antiparallel model of the TnI-TnC complex and a structural model derived from low-angle X-ray and neutron scattering studies. |
| doi_str_mv | 10.1023/A:1005352324741 |
| format | Article |
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In this work we extended the mapping of the TnC-TnI interface by analysing photocrosslinking between TnI and BP-Mal labelled TnC mutants with single Cys residues at positions 21 (TnC21) and 158 (TnC158). We determined the sites of these photocrosslinks in TnI by progressive proteolysis of the crosslinked product, followed by N-terminal sequencing and mass spectrophotometric analyses. The results show that whereas TnC158 forms a specific crosslink with Met-21, TnC21 forms multiple crosslinks in the range of residues 96 to 134 of TnI. 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In this work we extended the mapping of the TnC-TnI interface by analysing photocrosslinking between TnI and BP-Mal labelled TnC mutants with single Cys residues at positions 21 (TnC21) and 158 (TnC158). We determined the sites of these photocrosslinks in TnI by progressive proteolysis of the crosslinked product, followed by N-terminal sequencing and mass spectrophotometric analyses. The results show that whereas TnC158 forms a specific crosslink with Met-21, TnC21 forms multiple crosslinks in the range of residues 96 to 134 of TnI. 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| fulltext | fulltext |
| identifier | ISSN: 0142-4319 |
| ispartof | Journal of muscle research and cell motility, 1998-06, Vol.19 (5), p.479-490 |
| issn | 0142-4319 1573-2657 |
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| source | MEDLINE; SpringerLink Journals - AutoHoldings |
| subjects | Amino Acid Sequence Animals Benzophenones - metabolism Chromatography, High Pressure Liquid Cysteine - metabolism Maleimides - metabolism Models, Chemical Muscle, Skeletal - chemistry Mutation - genetics Neutrons Peptide Mapping Rabbits Spectrophotometry Structure-Activity Relationship Troponin C - chemistry Troponin C - genetics Troponin I - chemistry |
| title | Identification of the photocrosslinking sites in troponin-I with 4-maleimidobenzophenone labelled mutant troponin-Cs having single cysteines at positions 158 and 21 |
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