On the NMR analysis of pKa values in the unfolded state of proteins by extrapolation to zero denaturant.
Detailed knowledge of the pH-dependence in both folded and unfolded states of proteins is essential to understand the role of electrostatics in protein stability. The increasing number of natively disordered proteins constitutes an excellent source for the NMR analysis of pKa values in the unfolded state of proteins. However, the tendency of many natively disordered proteins to aggregate via intermolecular hydrophobic clusters limits their NMR analysis over a wide pH range. To assess whether the pKa values in natively disordered polypeptides can be extrapolated from NMR measurements in the presence of denaturants, the natively disordered backbone of the C-terminal fragment 75 to 105 of Human Thioredoxin was studied. First, assignments using triple resonance experiments were performed to confirm lack of secondary structure. Then the pH-dependence of the amides and carboxylate side chains of Glu residues (Glu88, Glu95, Glu98, and Glu103) in the pH range from 2.0 to 7.0 was monitored using 2D 1H15N HSQC and 3D C(CO)NH experiments, and the behavior of their amides and corresponding carboxyl groups was compared to confirm the absence of nonlocal interactions. Lastly, the effect of increasing dimethyl urea concentration on the pKa values of these Glu residues was monitored. The results indicate that: (i) the dispersion in the pKa of carboxyl groups and the pH midpoints of amides in Glu residues is about 0.5 pH units and 0.6 pH units, respectively; (ii) the backbone amides of the Glu residues exhibit pH midpoints which are within 0.2 pH units from those of their carboxylates; (iii) the addition of denaturant produces upshifts in the pKa values of Glu residues that are nearly independent of their position in the sequence; and (iv) these upshifts show a nonlinear behavior in denaturant concentration, complicating the extrapolation to zero denaturant. Nevertheless, the relative ordering of the pKa values of Glu residues is preserved over the whole range of denaturant concentrations indicating that measurements at high denaturant concentration (e.g. 4 M dimethyl urea) can yield a qualitatively correct ranking of the pKa of these residues in natively disordered proteins whose pH-dependence cannot be monitored directly by NMR.
Quijada
Jeniffer
J
Department of Chemistry, City College of New York, 138th Street and Convent Avenue, New York, NY 10031, USA.
López
Gary
G
Versace
Rodney
R
Ramírez
Luis
L
Tasayco
María Luisa
ML
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D018698:Glutamic Acid / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D055672:Static Electricity; D013879:Thioredoxins / Q000737:chemistry
D000577:Amides; D011506:Proteins; D018698:Glutamic Acid; D013879:Thioredoxins