Sites of Intra- and Intermolecular Cross-linking of the N-terminal Extension of Troponin I in Human Cardiac Whole Troponin Complex*
AUTOR(ES)
Warren, Chad M.
FONTE
American Society for Biochemistry and Molecular Biology
RESUMO
Our previous studies (Howarth, J. W., Meller, J., Solaro, R. J., Trewhella, J., and Rosevear, P. R. (2007) J. Mol. Biol. 373, 706–722) of the unique N-terminal region of human cardiac troponin I (hcTnI), predicted a possible intramolecular interaction near the basic inhibitory peptide. To explore this possibility, we generated single cysteine mutants (hcTnI-S5C and hcTnI-I19C), which were labeled with the hetero-bifunctional cross-linker benzophenone-4-maleimide. The labeled hcTnI was reconstituted to whole troponin and exposed to UV light to form cross-linked proteins. Reversed-phase high-performance liquid chromatography and SDS-PAGE indicated intra- and intermolecular cross-linking with hcTnC and hcTnT. Moreover, using tandem mass spectrometry and Edman sequencing, specific intramolecular sites of interaction were determined at position Met-154 (I19C mutant) and Met-155 (S5C mutant) of hcTnI and intermolecular interactions at positions Met-47 and Met-80 of hcTnC in all conditions. Even though specific intermolecular cross-linked sites did not differ, the relative abundance of cross-linking was altered. We also measured the Ca2+-dependent ATPase rate of reconstituted thin filament-myosin-S1 preparation regulated by either cross-linked or non-labeled troponin. Ca2+ regulation of the ATPase rate was lost when the Cys-5 hcTnI mutant was cross-linked in the absence of Ca2+, but only partially inhibited with Cys-19 cross-linking in either the presence or absence of Ca2+. This result indicates different functional effects of cross-linking to Met-154 and Met-155, which are located on different sides of the hcTnI switch peptide. Our data provide novel evidence identifying interactions of the hcTnI-N terminus with specific intra- and intermolecular sites.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2682874Documentos Relacionados
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