The binding site of rabbit skeletal α-tropomyosin on troponin-T

Abstract

The binding site for rabbit skeletal .alpha.-tropomyosin on troponin-T was localized to the CNBr fragment, CB2 (residues 71-151), using affinity chromatography. The entire fragment was required to bring about the correct secondary structure conducive to binding. CB2 contained about 80% .alpha.-helix and accounted for most of the helical content found in troponin-T (35%). MW CB2 obtained by sedimentation equilibrium (9700) agreed closely with the value calculated from sequence analysis (9850). Circular dichroism and sedimentation velocity experiments indicated that the helix was stable and not affected by salt concentrations of 0.1-1.6 M KCl nor by composition of the buffer. The helical content was unaffected by pH from 3.3-9.1 but decreased at pH 10-11. Temperature denaturation studies of CB2 and troponin-T showed that both were similarly heat labile, with loss of 50% of the ellipticity at 39.degree. C. Binding of CB2 to .alpha.-tropomyosin occurred in the pH range of 5.0-9.1, but not at pH 3-4 or 10-11. The helical region of CB2, and perhaps the carboxyl side chains of aspartic and glutamic acids, may be involved in binding over a limited surface area of the double-stranded coiled coil of .alpha.-tropomyosin.