Open AccessIdentification of an active site peptide of skeletal myosin after photoaffinity labeling with N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate.
Author(s) -
Yoh Okamoto,
Ralph G. Yount
Publication year - 1985
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.82.6.1575
Subject(s) - photoaffinity labeling , edman degradation , chemistry , peptide , myosin , active site , binding site , peptide sequence , amino acid , biochemistry , affinity label , stereochemistry , enzyme , gene
The active site of skeletal myosin has been photoaffinity labeled (approximately equal to 50%) by the ADP analog N-(4-azido-2-nitrophenyl)-2-aminoethyl triphosphate (NANDP) following the cobalt phenanthroline active site trapping procedure of Wells and Yount [Wells, J. A. & Yount, R. G. (1979) Proc. Natl. Acad. Sci. USA 76, 4966-4970]. Extensive proteolytic digestion of [3H]NANDP-labeled myosin subfragment one yielded two major peptides, P1 and P2, which were purified by reversed-phase high-performance liquid chromatography. These peptides represented 50% of all labeled amino acids and contained 1 mol of the unusual amino acid epsilon-N-trimethyllysine. Analysis of P2 by Edman techniques gave a sequence Val-Asn-Pro-Tyr-Lys(Me3)-X-Leu-Pro-Val-Tyr, which corresponds to an identical sequence for residues 125-134 determined by Tong and Elzinga [Tong, S. W. & Elzinga, M. (1983) J. Biol. Chem. 258, 13100-13110] for a segment of rabbit skeletal myosin heavy chain in which X is Trp-130. P1 was identical to P2 except it contained an additional three amino acids, Asn-Pro-Gln, at the COOH-terminal end. Amino acid composition, sequence data, spectral measurements, and location of radioactive label in both P1 and P2 all indicate Trp-130 is the major site of labeling by NANDP. The adjacent epsilon-N-trimethyllysine may provide part of the binding site for the triphosphate portion of ATP.