pKa Measurements from Nuclear Magnetic Resonance for the B1 and B2 Immunoglobulin G-Binding Domains of Protein G: Comparison with Calculated Values for Nuclear Magnetic Resonance and X-ray Structures

Publisher Website
Google Scholar
Abstract
Two-dimensional homo- and heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine pKa values for all of the acidic residues in the B1 and B2 immunoglobulin G- (IgG-) binding domains of protein G. Due to the stability of protein G over a wide pH range, estimates of ionization constants were also obtained for some basic residues. These experimentally determined ionization constants were compared with values calculated from both X-ray and NMR-derived structures of B1 and B2 using the UHBD algorithm [Antosiewicz, J., et al. (1994) J. Mol. Biol. 238, 415−436]. This algorithm has been found to be predictive for pKa measurements in proteins and, in combination with experimental measurements, allowed some evaluation of the NMR and X-ray structures. Three regions where significant differences exist between the X-ray and NMR structures are (1) the position of the E56 side chain relative to the backbone amides of K10 and D40, (2) residues 33−37 in the helix, and (3) the Y45 side-chain conformation. For all three cases, the experimental pH titration curves are notably more consistent with the X-ray structures than the NMR structures. In contrast, a number of solvent-accessible side chains have experimental pKas more in agreement with mean pKas calculated from families of NMR structures. The conformations of these side chains may be susceptible to crystal packing effects. From titration experiments under basic conditions, it is noteworthy that the chemical shift of the Y45 CεH resonance is invariant up to pDcorr 12. The Y45 side-chain hydroxyl group appears to maintain a nativelike hydrogen bond with D47 at pDcorr 12, even though the protein is approximately 90% unfolded. These results suggest that this short-range (i, i + 2) interaction, located in the β3−β4 hairpin, is present in the high-pH denatured state and may therefore form early in the folding of protein G.