Mapping Protein Structural Evolution upon Unfolding

In the past, many intensive attempts failed to capture or underestimated the copopulated intermediate conformers from the protein folding/unfolding reaction. We report a promising approach to kinetically trap, resolve, and quantify protein conformers that evolve during unfolding in solution. We conducted acid-induced unfolding of three model proteins (cytochrome c, myoglobin, and lysozyme), and the corresponding reaction aliquots upon decreasing the pH were electrosprayed for high field asymmetric waveform ion mobility spectrometry (FAIMS) measurements. The copopulated conformers were resolved, visualized, and quantified by a two-dimensional mapping of the FAIMS output. Contrary to expectations, all the above proteins appeared metamorphic (multiple-folded conformations) at the physiological pH, and cytochrome c exhibited an unusual "conformational shuttling" before forming the molten globule state. Thus, in contrast to many previous studies, a wide variety of thermodynamically stable intermediate conformers, including compact, molten globule, and partially unfolded forms, was trapped from solution, probing the unfolding mechanism in detail.