Detecting equilibrium cytochrome c folding intermediates by electrospray ionization mass spectrometry: Two partially folded forms populate the molten-globule state

Nanoelectrospray ionization mass spectrometry (nano-ESI-MS) is applied to the characterization of ferric cytochrome c (cyt c) conformational states under different solvent conditions. The methanol-induced molten-globule state in the pH range 2.6-3.0 is found to be populated by two distinct, partially folded conformers I-A and 1(B). The more compact intermediate I-B resembles that induced by glycerol in acid-unfolded cyt c. The less compact one, I-A, also can be induced by destabilization of the native structure by trifluoroethanol. I-A and 1(B) can be detected, in the absence of additives, around the midpoint of the acid-induced unfolding transition, providing direct evidence for involvement of equilibrium folding intermediates in cyt c conformational transitions at low pH. This study shows that mass spectrometry can contribute to the characterization of molten-globule states of proteins by detection of distinct, although poorly populated, conformations involved in a dynamic equilibrium.