Spatial proximity of the glycine-rich loop and the SH2 thiol in myosin subfragment 1.
Subfragment 1 (S1) prepared from rabbit skeletal muscle myosin was digested with trypsin to cleave the 95K heavy chain into three pieces, i.e., the 23K, 50K, and 20K fragments. The trypsin-treated S1 was then cross-linked with p-nitrophenyl iodoacetate. The cross-linker bridged one of the reactive thiols (SH2) in the 20K fragment and a lysine residue in the 23K fragment [Hiratsuka, T. (1987) Biochemistry 26, 3168-3173]. Location of the lysine residue was mapped along the 23K fragment by "end-label fingerprinting", which employed site-directed antibodies against the N-terminus of the 23K fragment and against the C-terminus of the 24K fragment (the 23K fragment plus nine extra residues at its C-terminus). The mapping revealed that Lys-184 or Lys-189 was the residue cross-linked with SH2. Since the cross-linker used here spans only several angstroms, the result indicates that Lys-184 or Lys-189 is very close to SH2 in the three-dimensional structure of myosin head. Examination of the primary structure of the 23K fragment has revealed that these lysine residues are in and very close to the so-called "glycine-rich loop", whose sequence is highly homologous to those of nucleotide-binding sites of various nucleotide-binding proteins.
Sutoh
K
K
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Hiratsuka
T
T
D000818:Animals; D003432:Cross-Linking Reagents; D005998:Glycine; D007461:Iodoacetates; D008970:Molecular Weight; D015879:Myosin Subfragments; D009218:Myosins; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D011817:Rabbits; D013438:Sulfhydryl Compounds; D014357:Trypsin
D003432:Cross-Linking Reagents; D007461:Iodoacetates; D015879:Myosin Subfragments; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; C053421:4-nitrophenyl iodoacetate; D014357:Trypsin; D009218:Myosins; D005998:Glycine