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

Abstract

Tropomyosin was labeled with a maleimide nitroxide spin-label attached to cysteine-190 via a succinimido ring which was subsequently opened by incubation at alkaline pH. Electron spin resonance (ESR) spectra showed a temperature-dependent equilibrium, below the main unfolding transition of tropomyosin, between labels which were restricted in their motion (strongly immobilized), predominating at low temperatures, and those which were highly mobile (weakly immobilized), predominating at higher temperatures. These label states were associated with two protein states from a comparison of the ESR spectral changes with the thermal unfolding profile of tropomyosin. The strongly immobilized labels were associated with the completely folded molded and the weakly immobilized labels with a partially unfolded (in the cysteine-190 region) state which is an intermediate in the thermal unfolding of tropomyosin. A spectral subtraction technique was used to measure the concentration ratio of strongly and weakly immobilized labels from which an equilibrium constant, K, was determined at different temperatures. A linear van't Hoff plot was obtained, indicating that the spin-labeled protein is in thermal equilibrium between these two conformational states with delta H = 17 kcal/mol, delta S = 56 cal/(deg X mol), and K = 1.0 at 34 degrees C. An upper limit of 10(7) s-1 for the conformational fluctuation was estimated from the shapes and separation of the two ESR spectral components. In contrast to the label with the opened succinimido ring, the spin-label with an intact succinimido ring remained strongly immobilized on the protein, indicating that in the partially unfolded state the molecule retains structure in the cysteine-190 region.

Unfolding domains of recombinant fusion αα‐tropomyosin

“…In contrast to calorimetric data, however, circular dichroism (CD) studies on striated tropomyosin only show one major unfolding transition (Woods, 1969;Lehrer, 1978;Holtzer et al, 1983) with a broad unfolding pretransition involving at least 20% of the a-helix (Woods, 1976;Betteridge & Lehrer, 1983). Fluorescence (Graceffa & Lehrer, 1980;Betteridge & Lehrer, 1983) spin label (Graceffa & Lehrer, 1984), and enzyme digestion studies (Ueno, 1984) suggest that the region that unfolds in the pretransition is approximately bounded by residues 130-190. In this study on a recombinant rat striated fusion a atropomyosin (fusion-tropomyosin), information on the location of regions in tropomyosin that cooperatively unfold was obtained. Fusion-tropomyosin contains an ad-1319 Y. Ishii et ai.…”

Evidence for interaction between smooth muscle tropomyosin and caldesmon

“…Tropomyosin and caldesmon were exhaustively dialyzed vs 2 mM Mops, 0.1 mM EDTA, pH 7.5, before viscosity measurements were performed. The concentration of tropomyosin was determined by the Lowry method using rabbit skeletal tropomyosin as a standard [23] and that of caldesmon was determined from the absorbance at 276 nm [14]. The reported molecular mass of a caldesmon polypeptide chain has ranged from 120 to 150 kDa [5,8,14,24,25].…”

.alpha.-Helix to random-coil transition of two-chain, coiled coils: experiments on the thermal denaturation of doubly crosslinked dimeric .beta.-tropomyosin

“…The first, which used K,Fe(CN), oxidation (Graceffa & Lehrer, 1984), was carried out as follows. With that value, simultaneous solution of the absorbance equations for C, gives Cross-linking of pp tropomyosin was carried out in two alternative ways by modifications of methods described by Lehrer.…”

“…In the first approach, experiments are interpreted in a physically ad hoc manner, usually in terms of putative stages of localized melting (Cohen & Szent-Gyorgyi, 1957;Noelken, 1962;Noelken & Holtzer, 1964;Woods, 1969;Halsey & Harrington, 1973;Chao & Holtzer, 1975;Lehrer, 1978;Williams & Swenson, 1981;Potekhin & Privalov, 1982;Graceffa & Lehrer, 1984). In the first approach, experiments are interpreted in a physically ad hoc manner, usually in terms of putative stages of localized melting (Cohen & Szent-Gyorgyi, 1957;Noelken, 1962;Noelken & Holtzer, 1964;Woods, 1969;Halsey & Harrington, 1973;Chao & Holtzer, 1975;Lehrer, 1978;Williams & Swenson, 1981;Potekhin & Privalov, 1982;Graceffa & Lehrer, 1984).…”