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

Abstract

Myosin subfragment 1 (S-1) with its two reactive cysteine groups crosslinked by N,N'-p-phenylenedimaleimide (pPDM), is shown to be a stable analogue of S-1 X ATP and S-1 X ADP X 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 X ATP or S-1 X ADP X Pi, whereas its affinity is 1/100th of that of S-1 X 5'-adenylyl imidodiphosphate and 1/1,000th of that of S-1 X ADP. pPDM-S-1 is also similar to S-1 X ATP and S-1 X ADP X Pi in that its binding to actin is not inhibited by troponin-tropomyosin. In contrast, the binding of S-1, S-1 X ADP, and S-1 X 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. This suggests that modifying S-1 with pPDM stabilizes a conformation which mimics that induced by the binding of ATP.