Mechanism of acid-induced folding of proteins

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Abstract
We have previously shown [Goto, Y., Calciano, L. J., and Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577] that .beta.-lactamase, cytochrome c, and apomyoglobin are maximally unfolded at pH 2 under conditions of low ionic strength, but a further decrease in pH, by increasing the concentration of HCl, refolds the proteins to the A state with properties similar to those of a molten globule state. To understand the mechanism of acid-induced refolding of protein structure, we studied the effects of various strong acids and their neutral salts on the acid-unfolded states of ferricytochrome c and apomyoglobin. The conformational transition of cytochrome c was monitored at 20.degree. C by using changes in the far-UV CD and in the Soret absorption at 394 nm, and that of apomyoglobin was monitored by changes in the far-UV CD. Various strong acids (i.e., sulfuric acid, perchloric acid, nitric acid, trichloroacetic acid, and trifluoroacetic acid) refolded the acid-unfolded cytochrome c and apomyoglobin to the A states as was the case with HCl. For both proteins neutral salts of these acids caused similar conformational transitions, confirming that the anions are responsible for bringing about the transition. The order of effectiveness of anions was shown to be ferricyanide > ferrocyanide > sulfate > trichloroacetate > thiocyanate > perchlorate > iodide > nitrate > trifluoroacetate > bromide > chloride. This series is similar to the electroselectivity series of anions toward the anion-exchange resins [Gregor, H. P., Belle, J., and Marcus, R. A. (1955) J. Am. Chem. Soc. 77, 2713-2719; Gjerde, D. T., Schmuchler, G., and Fritz, J.S. (1980) J. Chromatogr. 187, 35-45], showing that preferential binding of anions to the A states causes the conformational transitions.