Myosin lever arm orientation in muscle determined with high angular resolution using bifunctional spin labels

Area of Science:

• Biophysics
• Structural Biology
• Muscle Physiology

Background:

• High-resolution structural data for the myosin light chain domain (LCD) in vertebrate muscle under ambient conditions remains elusive with current techniques like X-ray crystallography, cryo-EM, and fluorescence polarization.
• Understanding LCD dynamics is crucial for elucidating the mechanisms of muscle contraction.

Purpose of the Study:

• To develop and apply a novel electron paramagnetic resonance (EPR) method for measuring the orientation of the myosin LCD in muscle fibers at high resolution.
• To investigate the conformational changes of the myosin lever arm during muscle contraction states (rigor and relaxed).

Main Methods:

• Engineered specific cysteine pairs in the myosin regulatory light chain (RLC) for site-directed labeling with a bifunctional spin label (BSL).
• Utilized EPR spectroscopy on oriented, demembranated muscle fibers with BSL-labeled RLC to determine angular distributions of the LCD.
• Analyzed EPR spectra to resolve ordered and disordered populations of the labeled helices.

Main Results:

• Achieved an angular resolution of 4° for LCD orientation measurements.
• Identified distinct ordered (9-11°) and disordered (>38°) populations of labeled helices in the rigor state, with the ordered population oriented perpendicular to the muscle fiber axis.
• Observed a shift to a more disordered orientational distribution upon ATP addition (relaxation).
• Detected a lever arm orientation approximately 33° different from predictions based on cryo-EM models of isolated myosin heads.

Conclusions:

• The study establishes the feasibility of using EPR with spin labeling for high-resolution detection of myosin LCD rotation in muscle fibers.
• The findings provide crucial insights into myosin lever arm orientation and dynamics during muscle contraction, augmenting and clarifying data from other techniques.
• The results challenge and refine existing models of muscle contraction mechanisms.