Application of H/D Exchange Mass Spectrometry to Extract Protein Folding Energies Of Staphylococcal Nuclease Mutants

摘要

Estimated (DELTA)G values for folding energies of several Staphylococcal mutants using the "Kinetic" method show clear correlation with the values obtained from tryptophan fluorescence studies, even though the absolute value is consistently high, see Figure 3A. Estimated m values are significantly lower than the fluorescence values and seem to get better for tightly folded proteins, Figure 3B. On the other hand the "Equilibrium" method produces (DELTA)G and m values agreeing well with fluorescence values (Figures 5 & 6). We tested the equilibrium method for two mutants having low and high folding energies along with the wild type. All are in excellent agreement with fluorescence values (Figure 6). Success of HX-ESI-MS kinetic method, like SUPREX, relies on an assumed HX mechanism, EX2. For fourteen mutants studied by ESI, HX-MS, higher resolution revealed two populations (closed & open) indicating the assumption of an HX mechanism (from prior MALDI studies) is not correct.