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 demonstrated that the unfolding and refolding of a large protein, rabbit muscle aldolase (M(r) 157 kDa), can be studied by combining amide hydrogen exchange 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 labeled with deuterium following a brief exposure to D(2)O. Electrospray ionization mass spectra of both the intact protein and its peptic fragments had multiple 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 and equilibrium constants describing the unfolding and refolding processes characteristic of aldolase destabilized in urea. The change in solvent-accessible surface, which has been used as a reaction coordinate for protein folding, is estimated from the dependence of the equilibrium and rate constants on the concentration of urea.
Deng
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Y
Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, USA.
Smith
D L
DL
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium / Q000737:chemistry; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011817:Rabbits; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013816:Thermodynamics; D014508:Urea
D011506:Proteins; D006859:Hydrogen; D014508:Urea; D003903:Deuterium; D005634:Fructose-Bisphosphate Aldolase