Using optical tweezers to relate the chemical and mechanical cross-bridge cycles

Abstract

In most current models of muscle contraction there are two translational steps, the working stroke, whereby an attached myosin cross-bridge moves relative to the actin filament, and the repriming step, in which the cross-bridge returns to its original orientation. The development of single molecule methods has allowed a more detailed investigation of the relationship of these mechanical steps to the underlying biochemistry. In the normal adenosine triphosphate cycle, myosinÁadenosine diphosphateÁphosphate (M Á ADP Á P i ) binds to actin and moves it by ca. 5 nm on average before the formation of the end product, the rigor actomyosin state. All the other product-like intermediate states tested were found to give no net movement indicating that M Á ADP Á P i alone binds in a pre-force state.Myosin states with bound, unhydrolysed nucleoside triphosphates also give no net movement, indicating that these must also bind in a post-force conformation and that the repriming, post-to pre-transition during the forward cycle must take place while the myosin is dissociated from actin. These observations fit in well with the structural model in which the working stroke is aligned to the opening of the switch 2 element of the ATPase site.

Dynamics of actomyosin interactions in relation to the cross-bridge cycle

“…This is not likely to be the major pathway, given the value of K 3a and the slightly more favourable binding of actin to the switch 2 open state. If 10% of the cross-bridges took that route, it would be just below the limits of detection of a reverse working stroke in optical trap experiments (Steffen et al 2003;Steffen & Sleep 2004).…”

Introduction

“…There are two translational steps in the actomyosin crossbridge cycle, the working stroke, whereby an attached myosin crossbridge moves relative to the actin filament, and the repriming step, in which the crossbridge returns to its original orientation. John Sleep reported on his investigation of crossbridge movement (using optical tweezers) at the single molecule level resulting from the binding to actin of a variety of myosin intermediate states produced by the binding of ATP analogues (Steffen & Sleep 2004). With the exception of MÁADPÁP i , all states that might be regarded as product-like give a working stroke of zero, from which he concludes that these all bind to actin in a way that cannot go through the power stroke because they are already at the end of the power stroke, i.e.…”

Acto-Myosin Cross-Bridge Stiffness Depends on the Nucleotide State of Myosin II

“…We probed the differences in the two myosin isoforms for these velocity-determining parameters using single-molecule analysis with the optical trapping method introduced by Finer et al 15 The experimental set up as described previously 16,17 is schematized in Figure 1A. An actin filament suspended and held taut between the two optically trapped beads interacted with the myosin immobilized on the nitrocellulose-coated glass bead.…”

“…The experimental set up as described previously , is schematized in Figure A. An actin filament suspended and held taut between the two optically trapped beads interacted with the myosin immobilized on the nitrocellulose-coated glass bead.…”