The cycle of muscle contraction and relaxation requires a signal (Ca2+ release through the ryanodine receptor Ca2+ channel), contraction (force generation through ATP hydrolysis of actomyosin), and relaxation (Ca2+ sequestration through SERCA). The Thomas lab uses biophysical methods to understand the structural biology of muscle function and how disruption via mutation, aging, and disease progression affects muscle function and regulation. My thesis work has focused on determining the structural changes associated with calmodulin (CaM) regulation of the calcium release channel ryanodine receptor (RyR) and regulatory light chain (RLC), another member of the EF-hand Ca2+-binding superfamily, which binds myosin. These proteins are both important for muscle regulation, share many dynamic and structural properties and both bind and form large protein complexes with their respective targets.
The focus of my thesis work has been on CaM regulation of the calcium release channel RyR, which is important because disrupted Ca2+ homeostasis has emerged as a key abnormality in numerous diseases, including: heart failure (HF), fatal ventricular arrhythmias, and Alzheimer’s disease. A major cause of deregulated intracellular Ca2+ levels is Ca2+ leak through the RyR, in the sarcoplasmic reticulum (SR). CaM is a small ubiquitous Ca2+ signaling protein that regulates numerous intracellular targets, including RyR, which CaM regulates in a Ca2+-dependent manner. CaM structure when bound to RyR is of great interest as more evidence suggests that disrupted CaM-RyR interactions play a key role in the cause and development of heart failure and certain arrhythmias. Clearly, there is a need to elucidate the structural factors that underlie CaM’s loss of regulatory capacity on RyR channel function. My thesis work aims to resolve CaM structure when bound to RyR and determine how Ca2+ and mutation effects the structural equilibrium when bound using fluorescence technology.
I hypothesized that the observed functional effects of CaM on RyR are caused by structural changes in the CaM-RyR complex, and that elucidation of these structure-function connections will be crucial in the analysis and treatment of heart disease. Using site-directed labeling with spectroscopic probes and time-resolved fluorescence resonance energy transfer (TR-FRET), I measured the conformation of CaM in complex with full-length RyR in its native environment. Interestingly, CaM adopts two conformations when bound to RyR and Ca2+ shifts the equilibrium to favor a more closed conformation. The distance and dynamics measured in the presence of full-length RyR are unique when compared to a short peptide fragment of the CaM binding site of RyR, which suggests CaM binds full-length RyR using sites outside of the canonical binding site. This is an exciting result because this is the first time CaM structure has been resolved in complex with RyR in solution and strongly suggests the structural model that has been used in the field to date does not accurately reflect the binding mode of CaM in the presence of the native full-length RyR. This work was recently published in Biophysical Journal, McCarthy et al. https://doi.org/10.1016/j.bpj.2020.01.010.
During the past year of my thesis work I took an interest in myosin’s role in muscle contraction and started a collaboration with a physics student, Yahor Savich, in the Thomas lab. Our work aims to define the structural elements important for myosin function in the light chain-binding region, the least resolved portion of myosin in high resolution structures of the actomyosin complex, using bifunctional paramagnetic probes placed on RLC. This work uses a new spin probe, which enhances the angular resolution of the structural elements of myosin by an order-of-magnitude. These experiments have been done using two different soluble myosin fragments as well as in native filaments. This work is currently in preparation.