N-Benzyl-P-Toluenesulfonamide (BTS) Traps the Myosin Head in a Conformation Associated with Strong Myosin Based Layer Lines and Weak, Rapidly Reversible Actin Binding even in the Absence of Nucleotide

Abstract

During its working cycle myosin proceeds through several structural changes coupled to changes in the state of the nucleotide. Details of the coupling between nucleotide state and myosin conformation are still unclear. Several lines of evidence suggest that for the same nucleotide state several conformations of the myosin head coexist while the state of the nucleotide may only determine their relative proportions (e.g., Xu et al., Biochemistry 2003; Nesmelov et al., Biophys J. 2008).We were particularly interested whether with ADP or without nucleotide, i.e., when myosin binds tightly to actin, we can find experimental interventions for which (1) strong myosin based layer lines (MLLs) can be seen in 2D-X-ray diffraction patterns as sign of the closed, pre-power stroke conformation, and (2) binding kinetics of myosin to actin can be determined by fiber stiffness. While we were unsuccessful with blebbistatin, we found N-Benzyl-p-toluenesulfonamide (BTS) to accumulate myosin heads, both without nucleotide and with ADP, in structural states that generate strong MLLs. While BTS had no effect on MLLs in the presence of ATP, with ADP or no nucleotide BTS generated MLLs as strong as seen with ATP at high temperature. This suggests that essentially all myosin heads can be trapped in a closed, pre-power stroke conformation by BTS even in the absence of nucleotide. Dependence of fiber stiffness on speed of applied stretches showed that nucleotide free myosin heads in the presence of BTS not only generate strong MLLs but also have much lower affinity for actin than seen without BTS and show rapid reversibility of binding to actin similar to that previously seen by us for weak binding states.