Amino-acid replacements in an internal region of tropomyosin alter the properties of the entire molecule

Two isoforms of lobster muscle tropomyosin, a fast muscle type, fTm, and a slow muscle type, sTm1, are identical except for 15 residues within the reg ion of amino acids 39-80, which corresponds to exon 2 of the tropomyosin ge nes of many phyla. Although the difference in the sequence does not include the terminal regions, the two isoforms are extremely different in viscosit y, which is a good measure of the head-to-tail interaction strength and sho uld be dependent on the conformation of the terminal 7-9 residues. To deter mine the influence of amino-acid replacements in the internal region on the overall conformation and the functional properties of the molecule, we com pared the physical properties of the two isoforms and their interactions wi th other proteins, such as actin and myosin subfragment 1 (S1). Limited pro teolysis by trypsin and chymotrypsin showed that sTm1 is more susceptible t han fTm at the sites outside the region with the replaced residues. Compare d with fTm, sTm1 showed higher viscosity, had a higher actin affinity, and inhibited acto-S1 ATPase to a greater extent. Finally, the binding isotherm of S1-ADP to actin-sTm1 is less sigmoidal than that to actin-fTm. These re sults indicate that the amino-acid replacements in the internal region alte r the conformation and the physical properties of the entire molecule as we ll as its interactions with actin and myosin.