Probing The Divalent Cation-binding Region Of The Myosin Regulatory Light Chain During Muscle Contraction Using EPR

Abstract

We are using electron paramagnetic spectroscopy (EPR) to determine whether the myosin light chain domain (LCD) rotates as a rigid unit during muscle contraction. To accomplish this we are making orientational measurements at selected positions in the LCD using site-directed spin labeling of single-cysteine mutants of RLC. In addition, we are using double-cysteine RLC mutants to measure distance changes using dipolar EPR. We created two RLC mutants that contain a single cysteine (I35C or A55C) in the helices of the helix-loop-helix of the rat ventricular RLC (vRLC) divalent cation-binding site. Starting with each single-cysteine mutant, a second cysteine was added in the opposite helix (I35C/L49C and A55C/A32C) for distance measurements. The four RLC mutants were labeled with the spin probe MTSL and separately reconstituted into rabbit psoas muscle fiber bundles. The EPR spectra of the labeled fiber bundles in rigor were sensitive to muscle fiber orientation, for both I35C- and A55C-RLC mutants, indicating that the spin labels were highly oriented at both sites, making them ideal for measuring orientational changes. For both single-cysteine RLC mutants, myosin in rigor showed two populations of oriented spin labels that became substantially disordered in relaxation, while contraction induced order in a small fraction of the spin labels. The EPR spectra of the fibers with double-cysteine RLC mutants showed that the spin labels were less sensitive to muscle orientation, so they are suitable for distance measurements with myofibrillar preparations at low temperatures. This work is supported by NIH grant AR052360 to OR and by the Minnesota Supercomputing Institute.