Crosslinked myosin subfragment 1: a stable analogue of the subfragment-1.ATP complex.

Abstract

Myosin [vertebrate] subfragment 1 (S-1) with its 2 reactive cysteine groups crosslinked by N,N''-p-phenylenedimaleimide (pPDM), is a stable analog of S-1.cntdot.ATP and S-1.cntdot.ADP.cntdot.Pi, the predominant complexes present during the steady state hydrolysis of ATP by S-1. pPDM-S-1 binds to actin with about twice the affinity of S-1.cntdot.ATP or S-1.cntdot.ADP.cntdot.Pi, whereas its affinity is 1/100th of that of S-1.cntdot.5''-adenylyl imidodiphosphate and 1/1000th of that of S-1.cntdot.ADP. pPDM-S-1 is also similar to S-1.cntdot.ATP and S-1.cntdot.ADP.cntdot.Pi in that its binding to actin is not inhibited by troponin-tropomyosin. The binding of S-1, S-1.cntdot.ADP and S-1.cntdot.5''-adenylyl imidodiphosphate to actin is markedly inhibited by troponin-tropomyosin in the absence of Ca2+ when actin is in large excess over S-1. Evidently, modifying S-1 with pPDM stabilizes a conformation which mimics that induced by the binding of ATP.