Photochemical cross-linking between native rabbit skeletal troponin C and benzoylbenzoyl-troponin I inhibitory peptide, residues 104-115.
The troponin I (TnI) inhibitory region (residues 104-115) was synthesized with alpha-14C-labeled Gly-104 and a covalently linked benzoylbenzoyl (BB) moiety at the N terminus to yield a photoactivatable radioactive peptide (BBIp). BBIp was cross-linked to rabbit skeletal muscle troponin C (TnC) to locate the binding site on TnC. The TnC/BBIp mixture was subjected to photolysis in aqueous buffer at pH 7.5 in the presence or absence of Ca2+. A covalent (1:1) cross-linked protein-peptide complex (TnC.BBIp) was isolated in both cases. The cross-linked complex was digested with trypsin, and the peptide fragments were separated by reversed-phase high performance liquid chromatography. The radioactive cross-linked peptide was isolated and further characterized by peptide sequencing and mass spectrometry before and after cyanogen bromide cleavage. The results indicated that Met-155 of TnC was cross-linked to the BB moiety of BBIp in either the presence or absence of Ca2+. The biological activity of both the BBIp peptide and the cross-linked TnC.BBIp complex was studied and a model of the TnC.inhibitory peptide complex was derived using molecular dynamic and energy minimization calculations.
Ngai
S M
SM
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Sönnichsen
F D
FD
Hodges
R S
RS
D000595:Amino Acid Sequence; D000818:Animals; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010777:Photochemistry; D010782:Photolysis; D011487:Protein Conformation; D011817:Rabbits; D014336:Troponin / Q000737:chemistry* / Q000378:metabolism; D019209:Troponin C; D019210:Troponin I
D009842:Oligopeptides; D010446:Peptide Fragments; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; C085462:benzoylbenzoyl-troponin I inhibitory peptide (104-115)