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Calcium binding to the N-domain of troponin C initiates a series of conformational changes that lead to muscle contraction. Calcium binding provides the free energy for a hydrophobic region in the core of N-domain to assume a more open configuration. Fluorescence measurements on a tryptophan mutant (F29W) show that a similar conformational change occurs in the absence of Ca2+ when the temperature is lowered under pressure. The conformation induced by subzero temperatures binds the hydrophobic probe bis-aminonaphthalene sulfonate, and the tryptophan has the same fluorescence lifetime (7 ns) as in the Ca2+-bound form. The decrease in volume (delta V = -25.4 ml/mol) corresponds to an increase in surface area. Thermodynamic measurements suggest an enthalpy-driven conformational change that leads to an intermediate with an exposed N-domain core and a high affinity for Ca2+.

The complete amino-acid sequence of rabbit skeletal troponin-T is reported. The protein consists of a single polypeptide chain of 259 amino acids; it has an acetylated amino terminus and a molecular weight of 30,503. The sequence was determined by manual and/or automated Edman degradation techniques on the six fragments obtained after cleavage with cyanogen bromide. The larger fragments were further digested with trypsin, chymotrypsin, alpha-lytic protease, thermolysin, or pepsin to obtain smaller fragments suitable for manual sequencing. About 50% of the residues are charged at neutral pH with highly acidic amino-terminal (residues 1-39) and highly basic carboxyl-terminal regions (residues 221-259). Predictions of secondary structure indicate 37% helical content with two long sections (residues 80-102 and 122-146) in that portion of the molecule implicated in binding to tropomyocin. Two of the three phosphorylated sites in the molecule are located at serine-1 and serine-149 or -150. The sequence about the latter site resembles somewhat the phosphorylase kinase phosphorylation sites in phosphorylase alpha and troponin-I.

Tropomyosin, extracted from the leg muscle of frogs that had been injected with [32P]orthophosphate, was fractionated into two components, alpha and beta, on a CM-cellulose column. Radioactivity was associated only with the alpha component. A single phosphorylation site was located at serine-283 (pentultimate at the COOH-terminal end) of the frog alpha tropomyosin. The same phosphorylated peptide was recovered in low yields from both rabbit skeletal alpha and cardiac tropomyosin. The presence of covalently bound phosphate in alpha tropomyosin and its absence in the beta component of rabbit skeletal muscle was suggested by 31P NMR spectroscopy. The amino acid sequences around the phosphorylation sites of frog and rabbit tropomyosin are identical. Because this sequence is not similar to any other known phosphorylation site in proteins, this indicates the existence of either specific kinase or phosphatase that can distinguish between alpha and beta tropomyosins. In a model proposed for the head-to-tail overlap of alpha tropomyosin molecules, one O-phosphoserine-283 residue could form a salt linkage with lysine-6 on one side of the overlap region and another with lysine-12 on the other side. This would predict a difference in the stability of polymers of phosphorylated and nonphosphorylated alphaalpha and alphabeta dimers of tropomyosin.

We have isolated a cDNA clone from a human fibroblast cDNA library that contains the entire protein-coding region of a 1.1-kilobase mRNA. This mRNA encodes a 284-amino acid tropomyosin, the primary structure of which most closely resembles smooth muscle tropomyosin. Thus, the expression of both 284-amino acid muscle-type and 247-amino acid non-muscle-type tropomyosins appears to be a normal feature of human non-muscle cells. We also present evidence to suggest that this cytoskeletal tropomyosin and a human skeletal muscle beta-tropomyosin are derived from a common structural gene by an alternative RNA splicing mechanism.

A tentative amino-acid sequence for the COOH-terminal half of rabbit skeletal tropomyosin is reported. These studies confirm our previous conclusions that this tropomyosin consists of several different but similar polypeptide chains. In the sequence, nonpolar residues occur in two series at intervals of seven residues. Amino-acid residues in series I are three residues on the NH(2)-terminal side of, and four residues on the COOH-terminal side of, residues in series II. The presence of occasional charged or ambivalent residues in the positions of series I or II does not lead to a disruption of this long-range pattern. The majority of residues located between the nonpolar residues are charged or polar amino acids. Two highly similar or identical alpha-helices with the reported sequence can be packed together in parallel in a coiled-coil structure. These may be in register or staggered by seven residues or some multiple of it. The observation that groups of small hydrophobic side chains appear to alternate with groups of bulky side chains suggests that a staggered arrangement of the two alpha-helices would maximize the regularity and hydrophobic interactions of the coiled-coil. Model building considerations show that this would occur with a stagger of 14 residues. Such an arrangement could account for the end-to-end aggregation of tropomyosin in solution, and in crystal and tactoid filaments. However, a structure in which the two polypeptides are in register cannot be ruled out.