Disparate Molecular Properties of Two Hypertrophic Cardiomyopathy-Associated cMyBP-C Mutants Reveal Distinct Pathogenic Mechanisms Beyond Haploinsufficiency.

: Hypertrophic cardiomyopathy (HCM) is a common genetic cardiac disorder marked by abnormal thickening of the left ventricular myocardium, often leading to arrhythmias and heart failure. Mutations in sarcomeric protein genes, particularly , which encodes cardiac myosin-binding protein C (cMyBP-C), are major contributors to HCM pathogenesis. This study aims to investigate the structural and functional effects of two HCM-associated missense mutations, p.S236G and p.E334K, located within the C0-C2 domains of cMyBP-C. : Following in silico analysis, a bacterial expression system was applied, enabling the discrete C0-C2 domains of wild-type (cMyBP-C) and mutant (cMyBP-C and cMyBP-C) cMyBP-C proteins to be expressed and purified as recombinant proteins. Structural and stability changes were assessed using circular dichroism (CD), differential scanning calorimetry (DSC), and chemical denaturation assays. Functional impact on actin binding was also evaluated in vitro. : CD analysis revealed altered secondary structure in both mutants compared to the wild-type protein. Thermal and chemical stability assays indicated increased stability in the cMyBP-C mutant, suggesting that it exhibits a more rigid conformation. This increased rigidity corresponded with a significant reduction in the actin-binding affinity relative to the wild-type protein. : Our findings demonstrate specific detrimental effects of the p.E334K mutation and underscore the importance of understanding the structural and functional consequences of HCM-associated mutations to assist the development of targeted therapeutic strategies.