Khare D, Alexander P, Antosiewicz J, Bryan P, Gilson M, Orban J
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville 20850, USA.
Biochemistry. 1997 Mar 25;36(12):3580-9. doi: 10.1021/bi9630927.
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 C epsilonH 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 beta3-beta4 hairpin, is present in the high-pH denatured state and may therefore form early in the folding of protein G.
二维同核和异核核磁共振（NMR）光谱法被用于测定蛋白G的B1和B2免疫球蛋白G（IgG）结合结构域中所有酸性残基的pKa值。由于蛋白G在很宽的pH范围内都具有稳定性，因此还获得了一些碱性残基的电离常数估计值。使用UHBD算法[Antosiewicz, J.,等人（1994年）《分子生物学杂志》238卷，415 - 436页]，将这些实验测定的电离常数与根据B1和B2的X射线结构以及NMR衍生结构计算得到的值进行了比较。已发现该算法可预测蛋白质中的pKa测量值，并且与实验测量值相结合，可以对NMR和X射线结构进行一些评估。X射线结构和NMR结构之间存在显著差异的三个区域是：（1）E56侧链相对于K10和D40主链酰胺的位置；（2）螺旋中的33 - 37位残基；（3）Y45侧链构象。对于所有这三种情况，实验pH滴定曲线与X射线结构的一致性明显高于与NMR结构的一致性。相比之下，一些溶剂可及的侧链的实验pKa值与根据NMR结构家族计算的平均pKa值更一致。这些侧链的构象可能易受晶体堆积效应的影响。从碱性条件下的滴定实验值得注意的是，直到pDcorr 12，Y45 CεH共振的化学位移都是不变的。即使蛋白质大约90%未折叠，在pDcorr 12时，Y45侧链羟基似乎仍与D47保持类似天然的氢键。这些结果表明，这种位于β3 - β4发夹结构中的短程（i，i + 2）相互作用存在于高pH变性状态，因此可能在蛋白G折叠的早期就形成。
