PK(A) 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

Two-dimensional homo- and heteronuclear nuclear magnetic resonance (NM R) spectroscopy was used to determine pK(a) values for all of the acid ic residues in the B1 and B2 immunoglobulin G-(IgG), binding domains o f protein G. Due to the stability of protein G over a wide pH range, e stimates of ionization constants were also obtained for some basic res idues. These experimentally determined ionization constants were compa red with values calculated from both X-ray and NMR-derived structures of Fl and B2 using the UHBD algorithm [Antosiewicz, J., et al. (1994) J. Mel. Biol. 238, 415-436]. This algorithm has been found to be predi ctive for pK(a) measurements in proteins and, in combination with expe rimental measurements, allowed some evaluation of the NMR and X-ray st ructures. Three regions where significant differences exist between th e 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 case s, the experimental pH titration curves are notably more consistent wi th the X-ray structures than the NMR structures. In contrast, a number of solvent-accessible side chains have experimental pK(a)s more in ag reement with mean pK(a)s calculated from families of NMR structures. T he conformations of these side chains may be susceptible to crystal pa cking effects. From titration experiments under basic conditions, it i s noteworthy that the chemical shift of the Y45 CepsilonH resonance is invariant up to pD(corr) 12. The Y45 side-chain hydroxyl group appear s to maintain a nativelike hydrogen bond with D47 at pD(corr) 12, even though the protein is approximately 90% unfolded. These results sugge st that this short-range (i, i + 2) interaction, located in the beta 3 -beta 4 hairpin, is present in the high-pH denatured state and may the refore form early in the folding of protein G.