Stability of Segments of Rabbit a-Tropomyosin

Abstract

Rabbit IX-tropomyosin was cleaved into two pieces at the cysteine residue of each chain. The products were separated by chromatography and characterized by amino acid analysis, molecular weight determination in benign and denaturing solvents, optical rotation and circular dichroism. When the cleavage reaction was carried out under mild conditions which preserve the two-chain structure there was considerable loss of IX-helix in each segment.Thermal stability studies, monitored by optical rotation and circular dichroism, showed that the transition temperature of the N-terminal fragment at pH 7·6 was approximately 17°C higher than that of the C-terminal fragment. In acid solutions there is little difference in the thermal stability of the two segments. The least stable part of the molecule is conchided to be between residues 133 and 205 and this includes the troponin-binding site. The relative stabilities found for segments of rabbit IX-tropomyosin differ from recent published conclusions and this may be a result of the different methods used to study the loss of the IX-helical conformation.The two tropomyosin fragments, unlike the parent tropomyosin, do not inhibit actomyosin adenosinetriphosphatase when mixed with troponin. The fragments did not show any of the aggregation properties of tropomyosin and did not combine with actin. The N-terminal fragment did not complex with troponin but there was some evidence for an interaction between the C-terminal fragment and troponin.

Long-Range Effects of Familial Hypertrophic Cardiomyopathy Mutations E180G and D175N on the Properties of Tropomyosin

“…Fluorescence probe studies with the label at Cys 190 have indicated that the probe responds to the unfolding in the pretransition temperature region (12, 13, 33). The main transition at 47°C is due to unfolding of the C-terminal region and the transition at about 54°C is due to unfolding in the N-terminal region (25, 26, 32). The relative areas under the derivative curves in Fig.…”

Structural Studies on the Microfibrillar Proteins of Wool: Characterization of the a-Helix-Rich Particle Produced by Chymotryptic Digestion

“…The length of coiled-coil regions is likely to be greater in the native keratin since the potential number of residues which can take up this conformation is 103 and 100 respectively for the two types of polypeptide chains involved (Fraser et al 1976) (~94 % ex-helix and a potential length of 15 nm). Fragments of the ex-helical muscle protein, a-tropomyosin, obtained by either chemical splitting or limited proteolysis (Woods 1977;Pato et al 1981) all show a lower ex-helix content than the parent protein. The molecular weight of 50200 for the helix-rich particle excludes the similar three subunit structure 'of Crewther and Dowling (1971), Crewther (1976) in favour of a four-chain particle.…”

Dynamic equilibrium between the two conformational states of spin-labeled tropomyosin

“…The molecule unfolds, with increasing temperature or concentration of denaturant, in two distinct stages (Woods, 1969;Pont & Woods, 1971;Satoh & Mihashi, 1972). The first stage, the pretransition, appears to involve unfolding of the C-terminal half of the molecule, and the second stage, the main transition, involves the complete unfolding of the molecule (Chao & Holtzer, 1975;Woods, 1977;Betcher-Lange & Lehrer, 1978;Lehrer, 1978;Pato & Smillie, 1978;Potekhin & Privalov, 1978). Woods (1976) has described this as an equilibrium between the native molecule, N, a partially unfolded (in the C-terminal half) intermediate state, X, and the completely denatured tropomyosin, D; Le., N F= X D. Studies of the fluorescence of probes attached to cysteine-190 (Graceffa & Lehrer, 1980;Betteridge & Lehrer, 1983), of the N M R of histidine-153 (Edwards & Sykes, 1980), and of differential scanning calorimetry of tropomyosin (Williams & Swenson, 1981;Potekhin & Privalov, 1982) have provided more direct evidence for multistate thermal equilibria.…”