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.

Myosin subfragment-2 (S2) is a coiled coil linker between myosin subfragment-1 and light meromyosin (LMM). This dissertation examines whether the myosin S2 coiled coil could regulate the amount of myosin S1 heads available to bind actin thin filaments by modulating the stability of its coiled coil. A stable myosin S2 coiled coil would have less active myosin S1 heads compared to a more flexible myosin S2 coiled coil, thus causing increased force production through acto-myosin interaction. The stability of the myosin S2 coiled coil was modulated by the binding of a natural myosin S2 binding protein, myosin binding protein C (MyBPC), and synthetic myosin S2 binding proteins, stabilizer and destabilizer peptide, to myosin S2. Competitive enzyme linked immunosorbent assay (cELISA) experiments revealed the cross specificity and high binding affinity of the synthetic peptides to the myosin S2 of human cardiac and rabbit skeletal origins. Gravitational force spectroscopy (GFS) was performed to test the stability of myosin S2 coiled coil in the presence of these myosin S2 binding proteins. GFS experiments demonstrated the stabilization of the myosin S2 coiled coil by the binding of MyBPC and stabilizer peptide to myosin S2, while the binding of destabilizer peptide to the same resulted in a flexible myosin S2 coiled coil. The binding of MyBPC and stabilizer peptide respectively, resulted in 3.35 and 1.5 times increase in force required to uncoil the myosin S2, while the binding of destabilizer peptide resulted in 1.6 times decrease in force required to uncoil the myosin S2. The myofibrillar contractility assay was performed to test the effect of synthetic myosin S2 binding proteins on the sarcomere shortening in myofibrils. The stabilizer peptide resulted in decreased sarcomere shortening of myofibrils as a result of decreased acto-myosin interaction, on the other hand, the binding of destabilizer peptide caused an increase in sarcomere shortening. The in vitro motility assay was performed to test the effect of altered stability of myosin S2 by binding of these myosin S2 binding proteins on the motility of actin filaments sliding over myosin. The motility of actin filaments was hindered by treating myosin thick filaments with whole length skeletal MyBPC or by treating heavy meromyosin with stabilizer peptide, while the motility of actin filaments was enhanced when heavy meromyosin was treated with destabilizer peptide. This study demonstrates that the myosin S2 coiled coil stability influences the force produced by acto-myosin interaction in striated skeletal muscle. The myosin S2 coiled coil when stabilized by MyBPC and stabilizer peptide resulted in decreased force production by reduced acto-myosin interaction. While the binding of destabilizer resulted in a flexible myosin S2 coiled coil and increased force production by enhanced acto-myosin interaction. The potentially cooperative response of contractility to the instability of the S2 coiled coil promises that this biological mechanism may be the target of drugs to modulate muscle performance.