Glutamic acid-88 is close to SH-1 in the tertiary structure of myosin subfragment 1.
The thiol-specific photoactivatable reagent benzophenone iodoacetamide (BPIA) can be selectively incorporated into the most reactive thiol, SH-1, of myosin S1, and upon photolysis, an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa region or with the central 50-kDa region [Lu et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392]. Comparison of the peptide maps of cross-linked and un-cross-linked S1 heavy chains indicates that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide [Sutoh & Lu (1987) Biochemistry 26, 4511]. In this report, S1 was labeled with radioactive BPIA, photolyzed in the absence of nucleotide, and then degraded with proteolytic enzymes. Peptides containing cross-links were isolated by liquid chromatography and subjected to amino acid sequence analyses. The results show that Glu-88 is the major site and Asp-89 and Met-92 are the minor sites involved in cross-linking with SH-1 (Cys-707) via BPIA. These residues are very near the reactive lysine residue (Lys-83) but relatively remote in the primary structure from the putative nucleotide binding region.
Lu
R C
RC
Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
Wong
A
A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001577:Benzophenones; D001665:Binding Sites; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D010450:Endopeptidases; D005971:Glutamates / Q000032:analysis*; D018698:Glutamic Acid; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D015879:Myosin Subfragments; D009218:Myosins / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis*; D010455:Peptides / Q000032:analysis; D010782:Photolysis; D011401:Promoter Regions, Genetic; D013438:Sulfhydryl Compounds / Q000032:analysis*
D000596:Amino Acids; D001577:Benzophenones; D005971:Glutamates; D015879:Myosin Subfragments; D010446:Peptide Fragments; D010455:Peptides; D013438:Sulfhydryl Compounds; D018698:Glutamic Acid; C049923:benzophenone-4-iodoacetamide; D010450:Endopeptidases; D009218:Myosins