Impact of Anti-S2 Peptides on a Variety of Muscle Myosin S2 Isoforms and Hypertrophic Cardiomyopathy Mutants Revealed by Fluorescence Resonance Energy Transfer and Gravitational Force Spectroscopy
Authors:   , Aboonasrshiraz Negar 
Published: February 2025

Myosin subfragment-2 (S2) is an intrinsically unstable coiled coil. This dissertation tests if the mechanical stability of myosin S2 would influence the availability of myosin S1 heads to actin thin filaments. The elevated instability in myosin S2 coiled coil could be one of the causes for hypercontractility in Familial Hypertrophic Cardiomyopathy (FHC). As hypothesized FHC mutations, namely E924K and E930del, in myosin S2 displayed an unstable myosin S2 coiled coil compared to wild type as measured by Fluorescence Resonant Energy Transfer (FRET) and gravitational force spectroscopy (GFS). To remedy this, anti-S2 peptides; the stabilizer and the destabilizer peptides by namesake were designed in our lab to increase and decrease the stability of myosin S2 coiled coil to influence the actomyosin interaction. Firstly, the effectiveness of anti-S2 peptides were tested on muscle myosin S2 peptides across MYH11 (smooth), MYH7 (cardiac), and MYH2 (skeletal) with GFS and FRET. The results demonstrated that the mechanical stability was increased by the stabilizer and decreased by the destabilizer across the cardiac and skeletal myosin S2 isoform but not for the smooth muscle isoform. The destabilizer peptide had dissociation binding constants of 9.97 × 10-1 μM to MYH7 isoform, 1.00 μM to MYH2 isoform, and no impact on MYH11, and the stabilizer peptide had dissociation binding constants of 2.12 × 10-2 μM to MYH7 isoform, 3.41 × 10-1 μM to MYH2 isoform, and no impact on MYH11 revealed by FRET. In presence of the stabilizer, FRET assay, affinity of the E930del and E924K increased by 10.23 and 0.60 fold respectively. The force required to uncoil muscle myosin S2 peptides in the presence of the stabilizer peptide was more than in its absence in muscle myosin S2 isoforms of MYH7 (1.80 fold higher), MYH2 (1.40 fold higher), and E930del (2.60 fold higher) and no change for MYH11 compared to control. The force required to uncoil muscle myosin S2 in presence of the destabilizer was less than in its absence in both MYH7 (2.00 fold lower) and MYH2 (2.5 fold lower) but the same for MYH11 compared to their controls. Both FRET and GFS assays demonstrated that both anti-S2 peptides do not have any impact on smooth muscle myosin S2 isoform. In FRET assay, there was no significant difference in the lifetime value in the presence or absence of anti-S2 peptides in smooth muscle myosin S2. In GFS assay, there was no significant difference in the force required to uncoil the dimer in presence or absence of the anti-S2 peptides smooth muscle myosin S2. Effectively, the stabilizer peptide improved the stability of FHC mutant (E924K and E930del) myosin S2 peptide. FHC mutations showed high lifetime value in FRET assay and low force to uncoil coiled coil myosin S2 in GFS assay. In the presence of the stabilizer, lifetime value decreased in FRET assay and more force was required to uncoil myosin S2 coiled coil in GFS assay. This study demonstrated that structure of muscle myosin S2 can be altered by small peptides. The stabilizer peptide enhanced dimer formation in wild type and mutant cardiac, and skeletal myosin S2 peptides, and destabilizer increased flexibility of cardiac and skeletal myosin S2 wild type peptide. Neither anti-S2 peptides had impacts on smooth muscle myosin S2 isoform. The study thus effectively demonstrates the mechanical stability of myosin S2 coiled coil in striated muscle system could be modified using the specific anti-S2 peptides. Stabilizer of the anti-S2 peptide was effective to remedy the dampened stability of FHC myosin S2 coiled coil thus providing a new dimension of treating cardiovascular and skeletal muscle disorders by targeting the structural property of muscle proteins.