Protein Equilibrium Population Snapshot (PEPS) MS method for Measuring Protein Folding Energies; H/D exchange vs Methionine Oxidation

摘要

Protein folding energies extracted using PEPS are clearly dependent upon the time that labeling proceeds. Shorter times are more consistent with NMR and fluorescence studies. As the labeling time increases, the number of folding/unfolding cycles experienced by proteins increases. In this situation, the extent of labeling will be perturbed by kinetic factors because, unlike fluorescence measurements, the oxidation reaction and H/D exchange are irreversible. For H/D exchange and methionine oxidation, it is clearly shown that the (DELTA)G_(app) vs [GdHCl] plots converge to accepted values at short labeling times. For both reaction probes, labeling times of less than 1 min were adequate as shown in figures 3 and 5. Ideally, probing processes hould be much faster than protein folding rates to allow the probing reaction to capture the unfolded state before it folds back within the fixed time interval. For example, tryptophan, tyrosine, and phenylalanine fluorescence (commonly used for this purpose) have fluorescence rates on the order of 10~(9) s~(-1). Although oxidation and H/D exchange rates are relatively slow compared to fluorescence rates, folding rates are drastically reduced at higher [GdHCl] so that K_(int)~(ox) and K_(int)~(H/D) K_(f).