According to current theory, force generation in muscle fibers is related to the conformational change that occurs as myosin, while hydrolyzing ATP, changes between its weakly- and strongly-binding configurations. Ever since we were the first to demonstrate the existence of weakly-binding crossbridges in muscle fibers, we have been interested in discerning the differences between weakly- and strongly-binding crossbridges. We have shown that one fundamental difference between weakly- and strongly-binding crossbridges is the ability of the former to maintain microsecond mobility while attached, whereas the latter are immobilized by attachment to actin. Most recently, we have been concerned with exploring what controls whether a crossbridge is in the weakly- or strongly-binding configuration, what controls the transition, and how this is related to the molecular mechanism of force generation. The state of myosin, as well as the ability to change state, seems to depend critically on a region of the myosin molecule near Cys-707 and Cys-697. Modifying these two sulfhydryls, which are known as SH1 and SH2, has dramatic effects on the states that myosin may attain. SH1 and SH2 both can be reacted either with 1 mole of paraphenylene dimaleimide (pPDM) or two moles of N-phenylmaleimide (NPM). Previously it was thought that both modifications lock myosin in a weakly-binding configuration. We investigated this in three ways; by studying the 2-dimensional X-ray diffraction patterns from muscle fibers in different types of rigor solution, by measuring the stiffness of muscle fibers both in the presence and absence of ATP, and by examining the strength of binding of myosin subfragment-1 to actin in solution. An important result was that myosin modified at SH1 and SH2 with NPM is capable of going into a strongly-binding configuration, even though that modified at SH1 and SH2 with pPDM is not. This suggests that a decrease in the flexibility of myosin in the region of SH1 and SH2 caused by covalent linkage of the two in the latter case may be an important factor in myosin's ability to go into the strongly-binding configuration. This has important implications for actomyosin's ability to generate force. Finally, preliminary results in FY97 suggest that EDC-crosslinked actomyosin may be a rather useful in vitro analogue for studying the in-vivo force-producing conformational changes.
