Flexibility of Human Cardiac α-Tropomyosin

α-Tropomyosin is the predominant tropomyosin isoform in adult human heart and constitutes a major component in Ca2+-regulated systolic contraction of cardiac muscle. Cardiac thin filaments are activated as Ca2+ induced conformational changes of the troponin complex lead to azimuthal movement of the tropomyosin strand to uncover myosin binding sites on the actin filament. The significance of tropomyosin flexibility in this process is highly speculated on, but an accurate measure of the protein's mechanical properties is not yet available. Changes in mechanical flexibility can also be an important mechanistic pathway towards altered functions and structural stability of α-tropomyosin observed in many familial hypertrophic cardiomyopathy mutants of the protein, such as that observed in the E180G mutant human cardiac α-tropomyosin. We present here the first direct probe images of wild type and E180G mutant human cardiac α-tropomyosin by atomic force microscopy. Mechanical flexibilities of both variants were quantified by three separate schemes of analysis. The persistence length of WT & α-tropomyosin equals 40 - 52 nm, corresponding to 1 - 1.3 molecular contour lengths. The E180G mutant was shown to be 34 - 37 % more flexible compared to the wild type. Corresponding to increased flexibility, we hypothesize that less mechanical moment, and hence a lesser extent of Ca2+ induced conformational change of troponin, are required to perturb α-tropomyosin to initiate thin filament activation during systole, leading to enhanced Ca2+-sensitivity. Hypersensitivity to Ca2+ could overwork cardiac muscle resulting in hypertrophic cardiomyopathy. We also demonstrated a sufficiently large population, with at least 100 molecules, is required for a reliable persistence length measurement of semi-flexible filamentous molecules, including α-tropomyosin. A separate experimental framework was also established to measure the mechanical flexibility of reconstituted thin filaments and yielded persistence length of actin filaments in close agreement with previous studies, which validates our methodology. The results presented in this dissertation extend our knowledge of the relationship between tropomyosin's mechanical properties and its regulatory function, as well as provide a mechanism between the E180G mutation of human cardiac α-tropomyosin and the associated functional changes of the protein. The experimental techniques and analysis framework developed here is readily applicable to polymerized α-tropomyosin, reconstituted thin filaments, as well as other disease related mutants of α-tropomyosin. This will provide a new avenue to further understand the role of α-tropomyosin in the mechanism of Ca2+-regulated activation of thin filament during muscle contraction.