Rate and equilibrium constants for protein unfolding and refolding determined by hydrogen exchange-mass spectrometry

Studies of protein unfolding and refolding may help us understand the more general problem of protein folding. Recent studies from this laboratory dem onstrated that the unfolding and refolding of a large protein, rabbit muscl e aldolase (M-r 157 kDa), can be studied by combining amide hydrogen exchan ge and mass spectrometry. Results of these studies indicated that aldolase has three unfolding domains which likely unfold sequentially. Urea was used to increase the populations of partially unfolded states which were labele d with deuterium following a brief exposure to D2O. Electrospray ionization mass spectra of both the intact protein and its peptic fragments had multi ple envelopes of isotope peaks from which the populations of unfolded forms were determined. The present study extends the previous investigations to include different urea concentrations and kinetic modeling of data taken as the system approaches equilibrium. Analysis of these results gives rate an d equilibrium constants describing the unfolding and refolding processes ch aracteristic of aldolase destabilized in urea. The change in solvent-access ible surface, which has been used as a reaction coordinate for protein fold ing, is estimated from the dependence of the equilibrium and rate constants on the concentration of urea. (C) 1999 Academic Press.