Studies of ligand-induced conformational perturbations in myosin subfragment 1. An examination of the environment about the SH2 and SH1 thiols using a photoprobe.
The effect of ligand binding on the environment near the SH2 and SH1 thiols in myosin subfragment 1 has been investigated by photocross-linking after specific labeling of these thiols individually with 4-(N-maleimido)benzophenone (MBP). On photolysis, cross-linking occurred from SH2-MBP to the middle 50-kDa segment, and subsequent immunopeptide mapping revealed that the cross-link was made to a peptide stretch 31-32 kDa from the N terminus in the absence of MgADP, whereas in its presence the cross-link occurred at about 60-61 kDa from the N terminus. Photolysis of SH1-MBP in the absence of MgADP resulted in a major cross-link to the 27-kDa N-terminal segment and minor cross-links to the 50-kDa middle segment. In the presence of MgADP, no new cross-link occurred but the amount of cross-linking to the 50-kDa segment increased at the expense of the other. Immunopeptide mapping indicated that the regions in the 27- and 50-kDa peptides that were cross-linked to SH1-MBP are at about 14-16 and 55-56 kDa from the N terminus respectively. These results indicate that when nucleotide binds to S1, SH2 is displaced relative to the 50-kDa segment, whereas the local environment around SH1 does not change significantly because photolysis in the presence of MgADP resulted in a change at the site of cross-linking for SH2-MBP but caused only a redistribution of the relative amounts of the cross-links formed from SH1-MBP.
Rajasekharan
K N
KN
Department of Biology, Case Institute of Technology, Case Western Reserve University, Cleveland, Ohio 44106.
Mayadevi
M
M
Burke
M
M
D000818:Animals; D001577:Benzophenones / Q000378:metabolism / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D008024:Ligands; D008301:Maleimides; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010782:Photolysis; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D014357:Trypsin
D001577:Benzophenones; D003432:Cross-Linking Reagents; D008024:Ligands; D008301:Maleimides; D015879:Myosin Subfragments; D010446:Peptide Fragments; C046109:benzophenone-4-maleimide; D014357:Trypsin; D009218:Myosins