Orientation of the N- and C-Terminal Lobes of the Myosin Regulatory Light Chain in Cardiac Muscle

Thomas Kampourakis,Yin-Biao Sun,Malcolm Irving

doi:10.1016/j.bpj.2014.11.049

Thomas Kampourakis, Yin-Biao Sun + Show 1 more

Open Access

https://doi.org/10.1016/j.bpj.2014.11.049

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Abstract

The orientations of the N- and C-terminal lobes of the cardiac isoform of the myosin regulatory light chain (cRLC) in the fully dephosphorylated state in ventricular trabeculae from rat heart were determined using polarized fluorescence from bifunctional sulforhodamine probes. cRLC mutants with one of eight pairs of surface-accessible cysteines were expressed, labeled with bifunctional sulforhodamine, and exchanged into demembranated trabeculae to replace some of the native cRLC. Polarized fluorescence data from the probes in each lobe were combined with RLC crystal structures to calculate the lobe orientation distribution with respect to the filament axis. The orientation distribution of the N-lobe had three distinct peaks (N1–N3) at similar angles in relaxation, isometric contraction, and rigor. The orientation distribution of the C-lobe had four peaks (C1–C4) in relaxation and isometric contraction, but only two of these (C2 and C4) remained in rigor. The N3 and C4 orientations are close to those of the corresponding RLC lobes in myosin head fragments bound to isolated actin filaments in the absence of ATP (in rigor), but also close to those of the pair of heads folded back against the filament surface in isolated thick filaments in the so-called J-motif conformation. The N1 and C1 orientations are close to those expected for actin-bound myosin heads with their light chain domains in a pre-powerstroke conformation. The N2 and C3 orientations have not been observed previously. The results show that the average change in orientation of the RLC region of the myosin heads on activation of cardiac muscle is small; the RLC regions of most heads remain in the same conformation as in relaxation. This suggests that the orientation of the dephosphorylated RLC region of myosin heads in cardiac muscle is primarily determined by an interaction with the thick filament surface.

Highlights

The globular head of the myosin molecule, called subfragment-1 or S1, is the molecular motor that drives muscle contraction, performing mechanical work in cyclical interactions with actin in the thin filament coupled to hydrolysis of ATP [1,2,3]
In most experiments bifunctional sulforhodamine (BSR)-cRLCs were introduced into skinned trabeculae from rat right ventricle using milder cRLC exchange conditions than those used previously in skeletal muscle [22,23,28], because recent results showed that the milder regulatory light chain (RLC) exchange protocol gives better preservation of thick filament structure [37]
In ventricular trabeculae this protocol resulted in replacement of ~12% of the native cRLC by BSR-cRLCs, which were mainly localized in the myosin-containing A-band of the sarcomere as expected (Fig. S2 A)

Summary

Introduction

The globular head of the myosin molecule, called subfragment-1 or S1, is the molecular motor that drives muscle contraction, performing mechanical work in cyclical interactions with actin in the thin filament coupled to hydrolysis of ATP [1,2,3]. The possibility that the functional lever arm might be composed of only part of the LCD, and that the functional pivot between the myosin motor and the thick filament might be within the LCD rather than at the S1-S2 junction, has not been excluded. This has fundamental implications for both molecular models of contractility and the regulatory role of the RLC in striated muscle

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