Thermal unfolding/refolding studies of the three tropomyosin dimers, alpha alpha, alpha beta, and beta beta, from chicken gizzard muscle were performed to explain the preferential assembly of alpha- and beta-tropomyosin subunits into heterodimers, alpha beta [Lehrer, S. S., & Qian, Y. (1989) J. Biol. Chem. 265, 1134]. Circular dichroism measurements showed that all three dimers unfolded in cooperative reversible transitions with T1/2 = 40.0 degrees C and delta H degrees = 162 kcal/mol for alpha alpha and with T1/2 = 42.6 degrees C and delta H degree = 98 kcal/mol for beta beta at 0.4-0.5 microM concentrations. Fluorescence measurements on pyrenyliodoacetamide-labeled tropomyosin showed that (i) excimer fluorescence decreases in parallel with unfolding of homodimers, (ii) at physiological temperature, heterodimers are formed from micromolar mixtures of homodimers over a period of minutes, and (iii) heterodimers unfold/refold with temperature without appreciable formation of homodimers. To understand the preferential formation of alpha beta, we calculated the concentrations of all species present as a function of temperature for equal total amounts of alpha and beta, using the measured thermodynamic constants of the unfolding/dissociation equilibria for alpha alpha and beta beta. Values for delta H degrees = 225 kcal/mol and T1/2 = 43 degrees C for unfolding of alpha beta at 0.5 microM concentration were obtained from the best fit of the calculations to the measured helical content vs temperature of alpha beta.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4366, 4367, 4368, 4369, 4370, 4371 Extra Details: additive : EDTA(1 mM),
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:25 p.m.
|Number of data points||9|
|Proteins||Tropomyosin alpha-1 chain ; Tropomyosin alpha-1 chain|
|Assays/Quantities/Protocols||Experimental Assay: dG ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence MDAIKKKMQMLKLDKENALDRAEQAEADKKAAEERSKQLEDELVALQKKLKGTEDELDKYSESLKDAQEKLELADKKATDC|