The acid-induced folded state of Sac7d is the native state

Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolde d protein is induced to refold by decreasing the pH to 0 ol by addition of salt (McCrary BS, Bedell J, Edmondson SP, Shriver JW, 1998, J Moi Biol 276: 203-224). Both near-ultraviolet circular dichroism spectra and ANS fluoresc ence enhancements indicate that the acid- and salt-induced folded states ha ve a native fold and are not molten globular. H-1,N-15 heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-ind uced folded states are essentially identical. The most significant differen ces in amide H-1 and N-15 chemical shifts are attributed to hydrogen bondin g to titrating carboxyl side chains and through-bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydroph obic core of the acid- and salt-induced folded states are identical to thos e observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the nativ e state. The folding of Sac7d as a function of pH and anion concentration i s summarized with a phase diagram that is similar to those observed for oth er proteins that undergo acid-induced folding except that the A-state is en compassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid.