The Histidine and Methionine Sequences of Rabbit Skeletal Tropomyosin

Tropomyosin, extracted from the leg muscle of frogs that had been injected with [32Plorthophosphate, was fractionated into two components, a and (3, on a CM-cellulose column. Radioactivity was associated only with the a component. A single phosphorylation site was located at serine-283 (penultimate at the COOH-terminal end) of the frog a tropomyosin. The same phosphorylated peptide was recovered in low yields from both rabbit skeletal a and cardiac tropomyosin. The presence of covalently bound phosphate in a tropomyosin and its absence in the % 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 a and P tropomyosins. In a model proposed for the head-to-tail overlap of a 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 ly-

Section: Methodsmentioning

confidence: 99%

“…Nucleic acid contamination was removed by chromatography on a DEAE-cellulose column as described by Woods (12). Carboxymethylation of the frog and rabbit tropomyosin was carried out as before (11 (13).…”

Section: Methodsmentioning

confidence: 99%

Specific phosphorylation at serine-283 of alpha tropomyosin from frog skeletal and rabbit skeletal and cardiac muscle.

Mak¹,

Smillie²,

Bárány³

1978

“…It is one of the components of the thin filaments of skeletal muscle, and is involved in the regulation of muscle contraction (1). Recently, considerable progress has been made in characterizing the two subunits (2) and in analyzing the amino-acid sequence (3)(4)(5), and it is clear that rabbit skeletal tropomyosin consists of two kinds of chains, a and fl, which differ slightly in amino-acid composition and sequence (2)(3)(4)(5). The a chain, which has one cysteine, is present in about a 3-fold excess over the ,3 chain, which contains two cysteines (2).…”

mentioning

confidence: 99%

Intramolecular crosslinking of tropomyosin via disulfide bond formation: evidence for chain register.

Lehrer¹

1975

170

121

The work reported below is an extension of these early observations using a different approach. We have found that it is possible to completely crosslink reduced tropomyosin by reaction with 5,5'-dithiobis(2-nitrobenzoate) (NbS)2. This aromatic disulfide causes the formation of S-S bonds between the corresponding cysteines of the two parallel chains within the same molecule by a two-step disulfide exchange process. Since this process requires the close proximity of two SH groups, these studies provide strong evidence for the model in which the chains are in register. In addition, from our consistent observations that only two crosslinked dimer chain species of approximately equal concentration are formed, it appears that the tropomyosin molecule is composed of only aa and a/3 chains.Tropomyosin is a rod-shaped molecule of 68,000 daltons whose subunits are made up of two parallel a-helical polypeptide chains of about equal molecular weight (1). It is one of the components of the thin filaments of skeletal muscle, and is involved in the regulation of muscle contraction (1). Recently, considerable progress has been made in characterizing the two subunits (2) and in analyzing the amino-acid sequence (3-5), and it is clear that rabbit skeletal tropomyosin consists of two kinds of chains, a and fl, which differ slightly in amino-acid composition and sequence (2-5). The a chain, which has one cysteine, is present in about a 3-fold excess over the ,3 chain, which contains two cysteines (2). Although the precise arrangement of chains is not known, there is some evidence to suggest that tropomyosin is not composed of a random mixture of a and /3 chains (6). Sodek et al. proposed a model for the interacting chains based on the observation that hydrophobic sidechains are predominantly located in two series which repeat every seventh residue in the sequence (7). This appears to allow maximum hydrophobic interaction along a line between the two a-helical chains when they are in register or when one chain is translated 7, 14, 21, ... residues with respect to the other. Although this model showed favorable contacts when the two chains were in register, the 14-residue staggered model was preferred because it appeared to optimize hydrophobic interactions and account for end-to-end aggregation.Evidence has accumulated over the years that unless care is taken to prevent heavy metal ion contamination, the SH groups of tropomyosin are readily oxidized to S-S (8-10). MATERIALS AND METHODSPreparation of Reduced Tropomyosin. Tropomyosin was prepared by the method of Bailey (11) as modified by Greaser and Gergely (12). Tropomyosin at about 5 mg/ml was dissolved in buffer (0.05 M Na phosphate, 0.01 M Na acetate, 1.0 M NaCl, 1 mM EDTA) at pH 7.5, containing 20 mM dithiothreitol, filtered through a Millipore HAWP 0.45 ,um filter, and warmed to 450 for 1 hr. After cooling, the pH was immediately reduced to 4.6 by addition of a few drops of HC1, and the isoelectric precipitate was collected by centrifugation for 15 min at 15,000 rpm. After di...

“…The open squares and circles represent two different series of hydrophobic residues, each of which repeats at every seventh residue in the aminoacid sequence. The residues of series I occur at positions 7,14,21, 28 …”

mentioning

confidence: 99%

Amino-Acid Sequence of Rabbit Skeletal Tropomyosin and Its Coiled-Coil Structure

Sodek

Hodges

Smillie

et al. 1972

Self Cite

162

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-2terminal side of, and four residues on the COOHterminal 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 longrange pattern. The majority of residues located between the nonpolar residues are charged or polar amino acids. Two highly similar or identical a-helices with the reported sequence can be packed together in parallel in a coiledcoil 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 a-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.Preparations of rabbit skeletal tropomyosin of molecularweight about 70,000 dissociate in denaturing medium to monomers of 35,000 daltons (1-6). The highly helical polypeptide chains are believed to be arranged in a two-stranded coiled-coil (7,8) or segmented rope (9) whose structure is stabilized by hydrophobic interactions between amino-acid residues situated in the core of the molecule. In such an arrangement, the regular interlocking of amino-acid side chains in a twostranded coiled-coil predicts a regular occurrence of alternating polar and nonpolar residues in tropomyosin. To test this prediction and to facilitate the interpretation of x-ray and electron microscopic studies of tropomyosin crystals and tactoids (10-12), we undertook the amino-acid sequence analysis of this protein. These studies (13-15) have demonstrated that, although the constituent polypeptide chains of preparations of rabbit skeletal tropomyosin are not chemically identical, they must be very similar in amino-acid sequence. Although it has not yet been possible to isolate these chains (of which there are at least four) in a pure state, it seemed feasible to perform a sequence analysis of the mixed population. Towards this end, cyanogen bromide cleavage of S-carboxymethylated tropomyosin was done, and a fragment with a minimum molecular weight of 17,000 was isolated by ion-exchange chromatography on QAE-Sephadex in 8 M urea (16,17). This fragment, which dimerizes to a molecular weight of 35,000 and assumes a relatively high degree of a-helical content (50-60%) in the abse...