[show abstract][hide abstract] ABSTRACT: Amino acid sequence analysis of the large cyanogen bromide fragment (residues 142 to 281) derived from the COOH-terminal half of the mixed tropomyosin population of rabbit skeletal muscle has been carried out. The isolation and sequence analysis of peptides derived from chymotryptic digests and from tryptic digests of the maleylated fragment permitted the alignment of the complete sequence except for the assignment of acids or amides at residues 142, 144, and 145. Selected peptides from a Myxobacter 495 alpha-lytic protease digest have confirmed certain overlaps. Based on previously published data the sequence can be extended to residue 284, the COOH-terminal end of the protein. In fourteen positions, amino acid substitutions have been observed. In one of these (residue 199) the sequence evidence indicates a minimum of four different polypeptide chains in the mixed tropomyosin population. The assignment of particular amino acid residues to these positions for the major alpha-component of rabbit skeletal tropomyosin has been based on the relative recoveries of peptides containing different residues in these positions.
Journal of Biological Chemistry 03/1978; 253(4):1129-36. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Previous studies have demonstrated that rabbit skeletal tropomyosin consists of two or more chemically non-identical but highly homologous polypeptide chains. Attempts by a variety of techniques to prepare pure tropomyosin chains in amounts adequate for chemical characterization have been unsuccessful to date. To provide more extensive information for the purpose of elucidating the relationship between amino acid sequence and the coiled-coil structure of tropomyosin, a cyanogen bromide treatment of the S-carboxymethylated protein was carried out. The fragments were separated into small and large components by gel filtration on Sephadex G-50. The small fragments were fractionated by ion-exchange chromatography and electrophoresis on paper and their sequences elucidated by conventional methods. Coupled with previous data, these results indicate a minimum of seven unique methionine sequences and are consistent with a high degree of homology in the tropomyosin polypeptide chains. From the mixture of the larger cyanogen bromide polypeptides, a fragment was isolated by ion-exchange chromatography on QAE-Sephadex. In aqueous buffer it had a molecular weight of 35 000 and an α-helical content of about 60% as estimated by circular dichroism. In 8 M urea its molecular weight was reduced to 15 000, a value in reasonable agreement with a minimal molecular weight of 17 000 calculated from its amino acid composition. From its histidine content (two residues) and the known COOH-terminal amino acid sequence of the protein, the fragment was concluded to be derived from the COOH-terminal half of the molecule. These results are consistent with a degree of 'coiled-coil' structure in a fragment representing about one-half of the tropomyosin molecule.
Canadian journal of biochemistry 02/1973; 51(1):56-70.
[show abstract][hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 01/1973; 69(12):3800-4. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Amino acid analyses of tropomyosin have shown three cysteine residues per mole (M.W. 70 000) of tropomyosin. The cysteine content was determined by cysteic acid determinations, incorporation of 14C-labelled iodoacetic acid into the protein, and the analysis of S-carboxymethylcysteine after acid hydrolysis. The isolation of three unique cysteinyl peptides is incompatible with a homogeneous tropomyosin preparation of two chemically identical subunits. The amino acid sequences reported in this study indicate a regular repeat of hydrophobic residues as required by the inter-chain packing of a coiled-coil structure.
Canadian journal of biochemistry 04/1972; 50(3):330-43.
[show abstract][hide abstract] ABSTRACT: Amino acid analyses of tropomyosin have previously shown four histidine and 13–14 methionine residues per mole (70 000 daltons) of tropomyosin. The isolation of two unique histidyl and five unique methionyl sequences is described. The number of unique methionyl peptides will undoubtedly be increased when more extensive sequence information becomes available although the value of 2 for the unique histidine sequences is considered to be a maximal one. These data support the conclusion that the two subunits of tropomyosin are similar in amino acid sequence. Both the acetylated NH2-terminal and COOH-terminal sequences of the protein have been determined in this study. The isolation and sequence analysis of two varieties of peptides arising from the COOH-terminus of the protein indicates either a degree of proteolysis during its isolation or a difference in the constituent polypeptide chains of tropomyosin in this region of their structures. The limited sequences reported indicate a repeat of hydrophobic residues as required by the inter-chain packing of a coiled-coil structure.
Canadian journal of biochemistry 04/1972; 50(3):312-29.
[show abstract][hide abstract] ABSTRACT: Amino acid analyses of tropomyosin have shown 4 histidines, 13–14 methionines and close to 3 cysteine residues per mole (M.Wt. 70,000) of tropomyosin. The isolation of 2 unique histidyl and 5 unique methionyl peptides is consistent with two identical polypeptide chains of molecular weight 35,000. However, the presence of 3 cysteines and 3 unique cysteine sequences per mole of 70,000 is indicative of either non-identical but similar chains or of more than one form of tropomyosin. This conclusion is further substantiated by the isolation and sequence analysis of two varieties of peptides arising from the C-terminus of the protein. The previous report of an N-acetylated peptide has been confirmed and its sequence elucidated. The limited sequences reported indicate a regular repeat of hydrophobic residues as required by the inter-chain packing of a coiled-coil structure.
Biochemical and Biophysical Research Communications 12/1970; 41(4):987-94. · 2.41 Impact Factor