Intradomain Distances in the Regulatory Domain of the Myosin Head in Prepower and Postpower Stroke States:  Fluorescence Energy Transfer

Abstract

The relative movement of the catalytic and regulatory domains of the myosin head (S1) is likely to be the force generating conformational change in the energy transduction of muscle [Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65]. To test this model we have measured, using frequency-modulated FRET, three distances between the catalytic domain and regulatory domains and within the regulatory domain of myosin. The donor/acceptor pairs included MHC cys707 and ELC cys177; ELC cys177 and RLC cys154; and ELC cys177 and gizzard RLC cys108. The IAEDANS (donor) or acceptor (DABMI or IAF) labeled light chains (ELC and RLC) were exchanged into monomeric myosin and the distances were measured in the putative prepower stroke states (in the presence of MgATP or ADP/AlF(4-)) and the postpower stroke states (ADP and the absence of nucleotides). For each of the three distances, the donor/acceptor pairs were reversed to minimize uncertainty in the distance measured, arising from probe orientational factors. The distances obtained from FRET were in close agreement with the distances in the crystal structure. Importantly, none of the measured distances varied by more than 2 A, putting a strong constraint on the extent of conformational changes within S1. The maximum axial movement of the distal part of myosin head was modeled using FRET distance changes within the myosin head reported here and previously. These models revealed an upper bound of 85 A for a swing of the regulatory domain with respect to the catalytic domain during the power stroke. Additionally, an upper bound of 22 A could be contributed to the power stroke by a reorientation of RLC with respect to the ELC during the power stroke.

Response of Rigor Cross-bridges to Stretch Detected by Fluorescence Lifetime Imaging Microscopy of Myosin Essential Light Chain in Skeletal Muscle Fibers

“…Observing a reduced fluorescence of fluorescein attached to the cysteine in chicken myosin subfragment-1 (S1) in the presence of ATP, Marsh et al (21) first proposed a change in the microenvironment of this residue. Further fluorescence resonance energy transfer studies produced contradictory results for the distance between the ELC and the catalytic domain (22,24). When energy transfer was measured in myosin S1, no significant change in distance was detected (24).…”

“…When energy transfer was measured in myosin S1, no significant change in distance was detected (24). However, a large change in distance between ELC and the catalytic domain, as well as between ELC and RLC, was reported when reconstituted myosin filaments were used (22). This contradiction could arise from differences of the in vitro systems used.…”

“…This residue has been exploited previously to attach fluorescent or spin probes (9,(21)(22)(23)(24). Observing a reduced fluorescence of fluorescein attached to the cysteine in chicken myosin subfragment-1 (S1) in the presence of ATP, Marsh et al (21) first proposed a change in the microenvironment of this residue.…”

The myosin power stroke

“…Due to its length and C-terminal location, it was proposed that this long ␣-helix might play a role as a lever arm that could amplify a small conformational change(s) generated in the core of the motor domain. Stimulated by this proposal, an array of observations from contracting fibers [Baker et al, 1998;Corrie et al, 1999;Irving et al, 1995], labeled subfragments in solution [Burmeister Getz et al, 1998;Palm et al, 1999;Shih et al, 2000;Suzuki et al, 1998;Xiao et al, 1998], tomographic reconstructions of insect flight muscle [Taylor et al, 1999], X-ray structures [Dominguez et al, 1998;Fisher et al, 1995;Houdusse et al, 2000;Smith and Rayment, 1996], and image reconstructions from cryo-EM data [Jontes et al, 1995; has subsequently provided support for the lever arm hypothesis.…”

Myosin binding protein C: Structural abnormalities in familial hypertrophic cardiomyopathy

“…Phosphorylation of cardiac MyBPC releases the S2 region, which is thought to allow the myosin crossbridges to reach out and interact more efficiently with actin, increasing force generation and systolic tension [48][49][50]. Flexibility of the myosin head, both as a whole, and within its two domains, is a necessary requirement for efficient force generation [51][52][53]. The extent of phosphorylation required to abolish the interaction with S2 remains unclear [4].…”