Cardiac Myosin-Binding Protein C N-Terminal Dynamics: Structural Insights from Time-Resolved Fluorescence Studies

Abstract

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated sarcomeric protein that modulates the strength and kinetics of cardiac muscle contraction and relaxation. This function is further mediated by PKA phosphorylation of cMyBP-C in response to beta-adrenergic stimulation. cMyBP-C is localized to the C-zone on each half of the sarcomere by interactions of its C-terminal domains with titin and myosin that anchor cMyBP-C to the thick filament backbone. The N-terminal domains of cMyBP-C may extend towards the thin filament, possibly to dynamically interact with myosin-thick filaments and/or actin-thin filaments in a phosphorylation-dependent manner. However, the molecular mechanisms responsible for cMyBP-C effects on cardiac muscle structure and function in normal physiological conditions remain incompletely understood. In an added level of complexity, mutations in the gene encoding cMyBP-C, MYBPC3, represent a leading cause of hypertrophic cardiomyopathy (HCM). A majority of cMyBP-C-associated HCM mutations are C-terminal truncation mutations leading to reduced cMyBP-C incorporation in the sarcomere (i.e., haploinsufficiency). In contrast, the molecular processes by which N-terminal cMyBP-C point (i.e., missense) mutations cause HCM are largely unknown. Therefore, the purpose of this Dissertation is to advance mechanistic knowledge, at the molecular level, of how N-terminal cMyBP-C structure and function are regulated by PKA-mediated phosphorylation and HCM mutations to influence myofilament function by using cutting-edge biochemical and biophysical approaches, including site-directed spectroscopy. Chapter 1 of this Dissertation provides a background reviewing known aspects of cMyBP-C structure and function in cardiac muscle contraction as well as an overview of site-directed spectroscopic methods. Chapter 2 examines cMyBP-C’s regulatory role in weak-to-strong transitions in actin structural dynamics in response to cMyBP-C binding and its phosphorylation as well as which domains modulate cMyBP-C’s cooperative effects on actin filament structural dynamics. Here, F-actin is labeled with a phosphorescent probe and time-resolved anisotropy (TPA) measurements resolve cMyBP-C N-terminal effects on the amplitudes and rates of actin filament twisting and bending. Chapter 3 identifies and characterizes a spectroscopy-based, phosphorylation- and mutation- sensitive cMyBP-C biosensor. Here, cMyBP-C N-terminal domains are labeled with donor-acceptor FRET (fluorescence resonance energy transfer) probes to monitor intramolecular distances and disorder due to phosphorylation, phosphomimetic mutation (Asp-for-Ser), and an HCM mutation affecting phosphorylation (Arg-282-Trp). Chapter 4 examines structural and functional perturbations in cMyBP-C N-terminal fragments and individual domains brought about by clinically-relevant HCM mutations on protein folding and function. Here, differential scanning calorimetry (DSC) as well as protein solubility and actin-binding assays are performed. Altogether, this Dissertation develops new structural biology-based research tools for mechanistic and therapeutic discovery and provides novel insight into the effects of cMyBP-C structure and function in response to post-translational modifications and disease-causing mutations.