Binding of SH1-SH2-modified myosin subfragment-1 to actin.
Myosin subfragment-1 (S1) was labeled with NPM in the presence of ATP or with pPDM in the presence of ADP at 0 degreesC, conditions which favor linking of maleimide groups to both Cys-707 (SH1) and Cys-697 (SH2). Unmodified S1 was removed by sedimentation with a small amount of F-actin, and the modified protein in the supernatant thoroughly dialyzed. The myosin high-salt EDTA and calcium ATPase activities of the isolated modified S1 were close to zero, suggesting nearly complete modification of SH1 and SH2. The binding of control and these modified myosins to actin was measured at 100 mM ionic strength using a co-sedimentation assay. In the presence of high MgATP concentration, control and NPM- and pPDM-reacted S1 all bind weakly to actin, with binding constants K3 of 4.9, 2.2, and 1.9 x 10(4) M-1, respectively. In the absence of MgATP, the binding constant K2 of pPDM-reacted S1 remains weak, 4.6 x 10(4) M-1,while that of NPM-reacted and control S1 becomes strong, 4.7 and 31 x 10(6) M-1, respectively. The binding constant for ATP to acto-NPM-reacted-S1 is approximately 2 x 10(4) M-1. Our data suggest that the binding of NPM-S1 to F-actin, in contrast to that of pPDM-S1, is ATP sensitive and can be quite strong at very low ATP concentration. They also suggest that while simple alkylation of SH1 and SH2 may be sufficient to inhibit myosin's ability to hydrolyze ATP, actual covalent linkage of SH1 and SH2 may be necessary to inhibit the weakly to strongly binding conformational change.
Xie
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Laboratory of Physical Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland 20892, USA.
Schoenberg
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M
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate; D000477:Alkylating Agents; D000818:Animals; D007700:Kinetics; D008301:Maleimides; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D013439:Sulfhydryl Reagents
D000199:Actins; D000477:Alkylating Agents; D008301:Maleimides; D015879:Myosin Subfragments; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D000255:Adenosine Triphosphate; C028525:N-phenylmaleimide; C011328:N,N'-4-phenylenedimaleimide