Transient Intermediary States with High and Low Folding Probabilities in the Apparent Two-state Folding Equilibrium of ACBP at Low pH

Measurements of the stability as a function of pH for the acyl-coenzyme A binding protein (ACBP) has shown a significant difference in the pH transition midpoint measured by NMR spectroscopy at pH 3.12 and the transition midpoint measured at pH 2.92 and 2.97 by circular dichroism and by fluorescence spectroscopy, respectively. A similar behavior has not been observed in other proteins. It is suggested that these differences arise because the population of the unfolded molecules still contains significant amounts of native like secondary and tertiary structure. NMR spectroscopy measures the concentration of the two components of the folding unfolding equilibrium individually, whereas circular dichroism and fluorescence measure the concentration of the conformations of the light-absorbing chromophores present in both the folded and the unfolded molecules. In the narrow pH range, nascent structure can be detected as the average amount of secondary structure per unfolded molecule and hydrophobic interactions in the population of unfolded molecules. These structures are not observable immediately by NMR spectroscopy; however, a chemical shift analysis of the peptide backbone 13C chemical shift indicates strongly the existence of short-lived and transient helical structures at pH 2.3.