The Conformational Stabilities of Tropomyosins

Abstract

The stability to denaturation by heat and guanidine hydrochloride of seven vertebrate (including skeletal, cardiac and smooth muscle) tropomyosins and three invertebrate tropomyosins was examined. The transition profiles were discontinuous and in many cases distinct plateaux were observed which indicated the presence of unique partially unfolded states at intermediate temperatures and guanidine hydrochloride concentrations.The denaturation by guanidine hydrochloride could be described in the majority of cases by a model in which the native state unfolds to a partially unfolded stable intermediate which then unfolds to the completely denatured state. On this basis it was possible to estimate the free energies of unfolding in water. It was shown that part of the IX-helical structure of tropomyosin is only marginally stable and the free energy of unfolding in water of this segment is less than values found for globular proteins, whereas another segment (or segments) has a stability comparable to that found for globular proteins. The stepwise unfolding may be explained in terms of the coiled-coil interactions in tropomyosin.Differences in stability were found between tropomyosins from different muscles of the same species as well as between species; no two tropomyosins giving the same denaturation profiles. The invertebrate tropomyosins showed a wider range of stabilities, that from scallop striated muscle being far more easily denatured than all the others. No correlation was found between the stability of tropomyosin and the type of regulatory system of the muscle. A comparison of the results from vertebrate and invertebrate species suggests that there has been no selection for proteins of higher or lower stability during the evolutionary time scale.

Stability of Segments of Rabbit a-Tropomyosin

“…Eckard and Cowgill (1976) have examined fragments produced by the tryptic digestion of rabbit tropomyosin and found that the C-terminal portion of the molecule was the most stable part. This is contrary to predictions on molecular stability deduced from the (X-helix-favouring potential of the amino acids (Woods 1976) and also contrary to some other experimental evidence (parry 1975). This paper reports the separation and characterization of fragments of (X-tropomyosin prepared by and Woods 1971).…”

“…He found that predictions on stability from the gross amino acid composition did not correlate with the observed stabilities to denaturation by heat and guanidine hydrochloride. The strength of the hydrophobic and electrostatic interactions between the two strands of the coiled-coil structure would need to be taken into account in order to predict the molecular stability (Woods 1976). The unstaggered coiled-coil structure (McLachlan and Stewart 1975) allows close packing of the non-polar side chains of the two helices and this arrangement also allows favourable interactions between positive and negative charges.…”

“…The mean residue weights were calculated from the amino acid compositions and a ).0 value of 212 nm was used. The temperature transitions were carried out as described in a previous paper (Woods 1976). Measurements of ORD at lower wavelengths and of circular dichroism (CD) were made with a Jasco instrument ORD/UV5 with a modified CD attachment and in some later experiments the temperature transitions were followed by the ellipticity at 222 nm, [6h22' or the mean residue rotation at the Cotton trough at 232 nm (m' 232).…”

“…The unfolding of tropomyosin by heat or guanidine hydrochloride has recently been explained by the existence of domains which differ sufficiently in stability to give separate stages in the denaturation profiles (Woods 1976). This model does not distinguish between two possibilities.…”

Characterisation of Fast, Slow and Cardiac Muscle Tropomyosins from Salmonid Fish

“…This need not involve a gross reduction in helical content. Evidence obtained using several techniques demonstrates that vertebrate striated muscle TMs undergo stepwise denaturation (Woods, 1976;Lehrer, 1978;Graceffa and Lehrer, 1980;Edwards and Sykes, 1980;Williams and Swenson, 1981 ;Potekhin and Privalov, 1982;Betteridge and Lehrer, 1983). A partial unfolding event, propagated through the coiled coil of the fast isoform to the overlap site (Edwards and Sykes, 1980), could account for the data collected at 25°C.…”