Tropomyosin: Long Range Perturbations In The Hydrophobic Interface

Abstract

Skeletal tropomyosin, (Tm), is an α-helical coiled-coil which binds to actin, and with troponin, regulates muscle contraction. We previously demonstrated that a conserved Asp 137 in the hydrophobic interface produces a dynamic region in the middle of Tm, and that this region is involved in the myosin dependent activation of the thin filament at high Ca2+, (Sumida et. al. JBC 283 2008). The current work characterizes a long-range interaction between positions 137 and 190. The thermodynamic properties of wild type (WT) Tm and two single mutants, C190A and D137L, are compared with those of the double mutant, D137L/C190A, using differential scanning calorimetry, (DSC), and circular dichroism, (CD). CD measurements show that Ala 190 increases the fraction of helix unfolding in the 40°C pre-transition, before the main transition. DSC measurements support this finding, indicating a large enthalpic pre-transition, (ΔH=150kcal/mol), for the C190A mutant relative to D137L, D137L/C190A, or WT, (average ΔH=20kcal/mol). Additionally, Ala 190 increases the ΔCp, (heat capacity), of Tm ∼5 fold, reflecting an increase in solvent exposure of hydrophobic residues in the pre-transition during unfolding. Since the D137L/C190A and D137L mutants do not exhibit the large enthalpic pre-transition observed for C190A, the Leu mutation at 137 must stabilize the alanine effect observed for C190A mutation 77Å away. This demonstrates how a locally dynamic region near 137 is able to produce global effects along the thin filament, and in this manner provide the proper regulation of the myosin dependent activation of the thin filament. This observation may also contribute to our understanding about the manner in which single point mutations significantly affect function in cardio-myopathies such as FHC and DCM.