The long-term goals of this research are to determine the role of the myosin light chain (LC) domain structural dynamics in the molecular mechanism of muscle contraction, and to understand how this is altered in muscle disease, focusing on LC mutations that cause familial hypertrophic cardiomyopathy (FHC). An innovative approach is proposed, using two complementary spectroscopic techniques: electron paramagnetic resonance (EPR) and fluorescence resonance energy transfer (FRET). Single and double cysteine mutations will be created on the LCs for site-directed labeling (SDL) with either spin or fluorescent probes. Most of these experiments will be performed on skinned muscle fibers, so that structural dynamics and function can be measured simultaneously on the same sample. We will test the hypothesis that the LC domain of myosin rotates as a unit during the powerstroke of muscle contraction, and that phosphorylation of the regulatory LC (RLC), divalent cation binding to RLC, and actin-binding of the essential LC (ELC) induce functionally important structural changes in this domain. We will also test the hypothesis that the mechanism for the FHC-LC disease is due to perturbations in the structural dynamics of the LC domain. We will use computational molecular dynamic simulations to test our results to develop models for muscle contraction.
AIM 1. Determine the angular changes of the myosin LC domain during muscle contraction. We will test the hypothesis that the myosin LC domain rotates as a unit in contracting muscle fibers. We will attach probes on site-directed cysteine mutants of RLC and ELC, then measure EPR and FRET.
AIM 2. Determine the effects of phosphorylation of RLC and divalent cation binding in RLC on the structural dynamics of myosin in muscle fibers. We will test the hypothesis that phosphorylation of RLC at Ser15 and binding of divalent cations induces dynamic changes in RLC with cysteine mutants using EPR and FRET.
AIM 3. Determine the role of the ELC in the structural dynamic interactions of myosin. We will use EPR and FRET to test the hypothesis that the N-terminal region of ELC interacts with the C-terminal region of actin in solution, and that the C-terminal domain of ELC interacts with the myosin heavy chain in muscle fibers.
AIM 4. Determine the effects of myosin LC FHC mutations on the structural dynamics and function of the LC domain in muscle fibers. Using the same approach as in the first three aims, we will test the hypothesis that LC FHC mutations induce functional and structural defects in myosin. This research, which combines novel biochemical preparations with state-of-the-art spectroscopic techniques, is designed to have a profound impact on our understanding of muscle structural dynamics, and to prepare the groundwork for translational research that applies results from basic muscle research to devise strategies for treating muscle disease.
