Fluorescence Based Approaches to Study Cam-Ryr Structural Interaction

Abstract

Excitation-contraction coupling in muscle is the physiological process of converting an electrical stimulus to a mechanical response. Release of Ca2+ from intracellular stores is essential for this process and is facilitated by calcium release from ryanodine receptor (RyR) calcium release channels. RyR channels are regulated by numerous small molecules and endogenous proteins, including calmodulin (CaM). CaM is a highly conserved, ubiquitously expressed, small dumb-bell shaped protein that binds four Ca2+ ions via four EF-hand motifs and regulates RyR in a calcium-dependent manner. At low (nM) [Ca2+] CaM is a partial agonist of RyR and it is an inhibitor at high (mM) [Ca2+]. The functional effects of CaM regulation of RyR are well established, but the structural mechanism of Ca2+-dependent regulation of RyR by CaM remains poorly understood. In part, this is due to the large size of RyR (2.2 MDa), which has limited most studies to peptide fragments of the CaM binding domain. The goal of this thesis is to elucidate the Ca2+-dependent conformational changes in CaM when in complex with full-length RyR, to gain insight into the mechanism of CaM-mediated regulation of RyR. Complementary fluorescence resonance energy transfer (FRET) and fluorescence-based stopped-flow kinetics experiments were performed to determine the Ca2+ -dependent structural changes in CaM when in complex with RyR. CaM was labeled with fluorescent probes in each lobe (N- and C-) and time-resolved FRET (TR-FRET) was used to assess inter-lobe distances (Chapter 3). With CaM bound to full-length RyR1, TR-FRET resolved two conformations, and Ca2+ stabilized a closed conformation by a factor of two. Surprisingly, an open conformation was the major component at high and low Ca2+, while the closed conformation was the major component in the presence of a peptide from the CaM binding domain of RyR 1. Calcium cycling in muscle contraction is a fast process, so to understand how Ca2+ binding events mediate conformational change in CaM when bound to full-length RyR1, CaM was labeled with an environment-sensitive probe and the rate of structural transition was monitored by stopped-flow kinetics (Chapter 4). We found differences in the rates of structural transition induced by Ca2+ binding between CaM bound to full-length RyR and a peptide from the CaM binding domain of RyR. These results provide new insights into the structural basis of CaM regulation of RyR1. CaM binds Ca2+ and undergoes structural transitions differently when bound to full-length RyR1 compared to the peptide. These differences may apply to other CaM targets and should motivate more structural work with CaM in the presence of full-length binding partners.