The stability of tropomyosin at acid pH: effects of anion binding

Abstract

The alpha-helical coiled-coil tropomyosin homodimer, alpha alphaTm, unfolds cooperatively with T1/2 = 47 degreesC, at neutral pH, 0.5 M NaCl. At pH 2, where each chain contains 55 positive charges, no cooperative unfolding occurs to at least 80 degreesC. The NaCl and K2SO4 dependence of thermal unfolding of alpha alphaTm and a less stable protein, beta betaTm, were studied with circular dichroism. For alpha alphaTm, 10 mM NaCl, or 0.5 mM K2SO4, was sufficient to increase the unfolding temperature by 80 degreesC. For beta betaTm, similar concentrations of NaCl and K2SO4 as for alpha alphaTm increased the unfolding temperature of the most stable domain. Titrations indicated that two to three anions bind preferentially to the beta betaTm intermediate. Thus anions bind to Tm at acid pH values to greatly stabilize the helix. But even in the absence of added salt, Tm is more stable at pH 2 than pH 7, suggesting destabilization by negatively charged amino acids.