SITE-DIRECTED SPECTROSCOPIC LABELING OF CARDIAC MYOSIN-BINDING PROTEIN C FOR INVESTIGATING PROTEIN STRUCTURAL DYNAMICS

Abstract

Cardiac myosin binding protein C (cMyBP-C) is a cardiac muscle protein that serves in the regulation of heart contractility. cMyBP-C mutations can be detrimental to health, promoting cardiac dysfunction and even heart failure. Particularly, abnormal cMyBP-C is associated with hypertrophy of the heart in a genetic condition known as familial hypertrophic cardiomyopathy. Currently, there is no cure for familial hypertrophic cardiomyopathy; treatment for the condition consists only of management of symptoms. Despite cMyBP-C’s association with hypertrophic cardiomyopathy, there still exists a void in our knowledge of cMyBP-C dynamics and functions. This is largely because of complexities due to size, disorder, and dynamics that arise when attempting to study it with conventional protein structural biology techniques. Therefore, my project aims to lay the foundation for studying cMyBP-C structure through the use of FRET spectroscopy, a technique that allows the measurement of intramolecular distances at the nanometer scale and structural disorder using fluorescent probe pairs. These probes are introduced at cysteine residues within cMyBP-C using thiol-reactive fluorescent dyes. Using this technique, we have encountered an issue in which our fluorescent dyes are labeling at greater amounts than the number of cysteines endogenous to human cMyBP-C allow for. Therefore, the purpose of this project was to study constructs of cMyBP-C with reduced numbers of endogenous cysteines to seek out the amino acid sites of cMyBP-C being unintentionally labeled, as well as identify improved labeling conditions to minimize observed overlabeling. We concluded that strategic substitution of cysteines with non-cysteine residues dramatically reduced labeling. Overlabeling is also reduced by lowering the pH of the cMyBP-C buffer during labeling. Lastly, FRET probe pairs in cMyBP-C reveal phosphorylation-dependent changes in N-terminal protein structure and dynamics that are important for its regulation of cardiac muscle.