Orientation of the N-Terminal Lobe of the Myosin Regulatory Light Chain in Skeletal Muscle Fibers

Romano, Daniela; Brandmeier, Birgit; Sun, Yin-Biao; Trentham, David R.; Irving, Malcolm

doi:10.1016/j.bpj.2012.02.010

Cited by 9 publications

(30 citation statements)

References 35 publications

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“…2A). We found that smRLC, used in previous studies , reports similar orientation compared to skRLC, consistent with previous results (Romano et al, 2012).…”

Section: Summary Of Resultssupporting

confidence: 92%

“…Fig. 2B shows that orientations of smRLC and skRLC are hardly distinguishable, which is consistent with fluorescence polarization experiments (Romano et al, 2012). Therefore, to maximize the physiological relevance of our work, we focused on skRLC from here on.…”

Section: Detecting Anisotropy In the Epr Fiber Experiments And Rlc Homsupporting

confidence: 85%

“…The above-mentioned rotation puts of the B and E helix to 14.6º and 85.3º, respectively. Thus, the result of (Romano et al, 2012) lies within the confidence interval of our method, whereas orientation of the B helix differs from ours by 10.3º. Even though fluorescence anisotropy experiments are versatile in measuring changes of orientation with high temporal resolution, one should not forget that maximum entropy distribution is a low-resolution approximation of the actual angular distribution, thus the difference in 10.3 o could be attributed to multiple factors.…”

Section: E Helix G103c-v107csupporting

confidence: 65%

“…To better contextualize our modeling results, we made comparisons with recent fluorescence anisotropy measurements (Romano et al, 2012). In that previous work, the authors defined a "lever axis" as a vector joining the αcarbons of Cys707 and Lys843 of the 2MYS model.…”

Section: E Helix G103c-v107cmentioning

confidence: 99%

“…In addition, none of the above-mentioned structural techniques provide structural information under physiological conditions (not frozen or crystallized) in muscle fibers. Fluorescence polarization does not have this limitation, and has been used to provide some orientational information about the ELC (Knowles et al, 2008) and RLC (Brack et al, 2004;Romano et al, 2012;Fusi et al, 2015) in skinned fibers. However, fluorescence has low angular resolution, so this approach requires data from multiple labeling sites and complex data analysis.…”

Section: Introductionmentioning

confidence: 99%

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Myosin Orientation in a Muscle Fiber Determined with High Angular Resolution Using Bifunctional Spin Labels

Savich

Binder

Thompson

et al. 2019

Preprint

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We used electron paramagnetic resonance to determine the orientation of elements within the myosin regulatory light chain in skinned skeletal muscle fibers. A bifunctional spin label provided sufficient resolution to detect an ordered population of lever arms perpendicular to actin.ABSTRACT We have measured the orientation of the myosin light chain domain in demembranated muscle fibers by electron paramagnetic resonance (EPR), using a bifunctional spin label (BSL), with angular resolution of 4 degrees. Despite advances in X-ray crystallography, cryoelectron microscopy (cryo-EM), and fluorescence polarization, none of these techniques provide highresolution structural information about the myosin light chain domain under ambient conditions in fiber. Two cysteines, 4 residues apart, were engineered on an α helix in the myosin regulatory light chain (RLC), permitting stereoselective site-directed labeling with BSL. By exchanging BSL-labeled RLC onto oriented muscle fibers, we obtained EPR spectra that resolved angular distributions in several biochemical states. The accurate determination of BSL's angular coordinates allowed us to determine the orientation of individual structural elements with respect to the muscle fiber axis. The addition of ATP, in the absence of Ca 2+ , shifted the orientation to a much more disordered distribution. We resolved an oriented population with lever arm oriented perpendicular to the muscle fiber axis, a state previously observed directly in insect flight muscle, but not in fibers having isolated myosin heads (S1) attached to actin.

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“…2A). We found that smRLC, used in previous studies , reports similar orientation compared to skRLC, consistent with previous results (Romano et al, 2012).…”

Section: Summary Of Resultssupporting

confidence: 92%

Section: Detecting Anisotropy In the Epr Fiber Experiments And Rlc Homsupporting

confidence: 85%

Section: E Helix G103c-v107csupporting

confidence: 65%

Section: E Helix G103c-v107cmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Myosin Orientation in a Muscle Fiber Determined with High Angular Resolution Using Bifunctional Spin Labels

Savich

Binder

Thompson

et al. 2019

Preprint

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Stiffness, working stroke, and force of single-myosin molecules in skeletal muscle: elucidation of these mechanical properties via nonlinear elasticity evaluation

Kaya

Higuchi

2013

Cell. Mol. Life Sci.

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In muscles, the arrays of skeletal myosin molecules interact with actin filaments and continuously generate force at various contraction speeds. Therefore, it is crucial for myosin molecules to generate force collectively and minimize the interference between individual myosin molecules. Knowledge of the elasticity of myosin molecules is crucial for understanding the molecular mechanisms of muscle contractions because elasticity directly affects the working and drag (resistance) force generation when myosin molecules are positively or negatively strained. The working stroke distance is also an important mechanical property necessary for elucidation of the thermodynamic efficiency of muscle contractions at the molecular level. In this review, we focus on these mechanical properties obtained from single-fiber and single-molecule studies and discuss recent findings associated with these mechanical properties. We also discuss the potential molecular mechanisms associated with reduction of the drag effect caused by negatively strained myosin molecules.

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

Kampourakis

Sun

Irving

2015

Biophysical Journal

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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.

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Orientation of the N-Terminal Lobe of the Myosin Regulatory Light Chain in Skeletal Muscle Fibers

Cited by 9 publications

References 35 publications

Myosin Orientation in a Muscle Fiber Determined with High Angular Resolution Using Bifunctional Spin Labels

Myosin Orientation in a Muscle Fiber Determined with High Angular Resolution Using Bifunctional Spin Labels

Stiffness, working stroke, and force of single-myosin molecules in skeletal muscle: elucidation of these mechanical properties via nonlinear elasticity evaluation

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

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