SKELETAL MYOSIN BINDING PROTEIN-C: INVESTIGATING THE EFFECTS OF DISTAL ARTHROGRYPOSIS MUTATIONS ON ACTIN BINDING

Abstract

Myosin-binding protein-C (MyBP-C) is a thick-filament associated protein involved in regulating muscle contraction through interactions with actin and myosin. MyBP-C consists of a family of protein isoforms, including fast skeletal MyBP-C (fMyBP-C), slow skeletal MyBP-C (sMyBP-C), and cardiac MyBP-C (cMyBP-C). sMyBP-C can be further categorized into various splice variants, including the long and short forms, due to post-translational alternative splicing. As another notable post translational modification, long sMyBP-C contains a protein kinase A (PKA)-mediated phosphorylation site that is absent in short sMyBP-C. Relevant to human disease, mutations in the gene encoding sMyBP-C have been shown to cause Distal Arthrogryposis (DA), a skeletal muscle disorder characterized by contractures affecting the distal joints of the wrists and ankles. The mutations W236R, P319L, and E359K have all been identified as causing DA, but the molecular mechanism behind this pathophysiology is still unknown. In efforts to understand more about this mechanism, these three DA mutants were introduced into the coding sequences of DNA for human short sMyBP-C N-terminal domains C1 through C2 (C1-C2) using site-directed mutagenesis by PCR. The resulting mutant proteins were expressed in E. coli bacterial cultures and purified via His-tag affinity chromatography. The mutations did not significantly impact protein folding and stability, as indicated by the results of protein solubility tests. Additionally, actin co-sedimentation assays showed that the DA mutations did not change the actin binding affinity of short C1-C2 in a statistically significant way. This same assay showed that short C1-C2 and long C1C2 similarly bind actin. Overall, this study provides biochemical insight into the functional impacts of DA mutants on the physiological and pathophysiological interactions between short sMyBP-C and its binding partner, actin, in the skeletal muscle sarcomere.