Accuracy of SUPREX (stability of unpurified proteins from rates of H/D exchange) and MALDI mass spectrometry-derived protein unfolding free energies determined under non-EX2 exchange conditions.
Described here is the impact of so-called non-EX2 exchange behavior on the accuracy of protein unfolding free energies (i.e., DeltaG u values) and m values (i.e.,-deltaDeltaG u/delta[denaturant] values) determined by an H/D exchange and mass spectrometry-based technique termed stability of unpurified proteins from rates of H/D exchange (SUPREX). Both experimental and theoretical results on a model protein, ubiquitin, reveal that reasonably accurate thermodynamic parameters for its folding reaction can be determined by SUPREX even when H/D exchange data is collected in a non-EX2 regime. Not surprisingly, the theoretical results reported here on a series of hypothetical protein systems with a wide range of biophysical properties show that the accuracy of SUPREX-derived DeltaG u and m values is compromised for many proteins when analyses are performed at high pH (e.g., pH 9) and for selected proteins with specific biophysical parameters (e.g., slow folding rates) when analyses are performed at lower pH. Of more significance is that the experimental and theoretical results reveal a means by which problems with non-EX2 exchange behavior can be detected in the SUPREX experiment without prior knowledge of the protein's biophysical properties. The results of this work also reveal that such problems with non-EX2 exchange behavior can generally be minimized if appropriate H/D exchange times are employed in the SUPREX experiment to yield SUPREX curve transition midpoints at chemical denaturant concentrations less than 2 M.
Dai
Susie Y
SY
Department of Chemistry, Duke University, Box 90346, 27708-0346, Durham, NC, USA.
Fitzgerald
Michael C
MC
Department of Chemistry, Duke University, Box 90346, 27708-0346, Durham, NC, USA. michael.c.fitzgerald@duke.edu.
D041961:Deuterium Exchange Measurement / Q000379:methods*; D006859:Hydrogen / Q000737:chemistry*; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry
D011506:Proteins; D025801:Ubiquitin; D006859:Hydrogen