Apparent pKa shifts of titratable residues at high denaturant concentration and the impact on protein stability.
Urea and guanidine-hydrochloride (GdnHCl) are frequently used for protein denaturation in order to determine the Gibbs free energy of folding and kinetic folding/unfolding parameters. Constant pH value is applied in the folding/unfolding experiments at different denaturant concentrations and steady protonation state of titratable groups is assumed in the folded and unfolded protein, respectively. The apparent side-chain pKa values of Asp, Glu, His and Lys in the absence and presence of 6 M urea and GdnHCl, respectively, have been determined by 1H-NMR. pKa values of all four residues are up-shifted by 0.3-0.5 pH units in presence of 6 M urea by comparison with pKa values of the residues dissolved in water. In the presence of 6 M GdnHCl, pKa values are down-shifted by 0.2-0.3 pH units in the case of acidic and up-shifted by 0.3-0.5 pH units in the case of basic residues. Shifted pKa values in the presence of denaturant may have a pronounced effect on the outcome of the protein stability obtained from denaturant unfolding experiments.
Marti
Daniel N
DN
Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. dmarti@access.unizh.ch
D001224:Aspartic Acid / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D018698:Glutamic Acid / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D017186:Titrimetry / Q000379:methods; D014508:Urea / Q000737:chemistry
D011506:Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D014508:Urea; D019791:Guanidine; D008239:Lysine