IDENTIFICATION OF THE PHOTO-CROSS-LINKING SITES IN TROPONIN-I WITH 4-MALEIMIDOBENZOPHENONE LABELED MUTANT TROPONIN-CS HAVING SINGLE CYSTEINES AT POSITION-158 AND POSITION-21

Our previous studies have shown that 3-maleimidobenzophenone (BP-Mal) attached to troponin-C (TnC) mutants with single cysteines at position s 12, 57, 89 and 98 forms crosslinks to troponin-I (TnI), and the iden tified 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-M al labelled TnC mutants with single Cys residues at positions 21 (TnC2 1) 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 re sults 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-ra y and neutron scattering studies. (C) Chapman & Hall Ltd.