Koide A, Jordan M R, Horner S R, Batori V, Koide S
Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, New York 14642, USA.
Biochemistry. 2001 Aug 28;40(34):10326-33. doi: 10.1021/bi010916y.
It is generally considered that electrostatic interactions on the protein surface, such as ion pairs, contribute little to protein stability, although they may play important roles in conformational specificity. We found that the tenth fibronectin type III domain of human fibronectin (FNfn10) is more stable at acidic pH than neutral pH, with an apparent midpoint of transition near pH 4. Determination of pK(a)'s for all the side chain carboxyl groups of Asp and Glu residues revealed that Asp 23 and Glu 9 have an upshifted pK(a). These residues and Asp 7 form a negatively charged patch on the surface of FNfn10, with Asp 7 centrally located between Asp 23 and Glu 9, suggesting repulsive electrostatic interactions among these residues at neutral pH. Mutant proteins, D7N and D7K, in which Asp 7 was replaced with Asn and Lys, respectively, exhibited a modest but significant increase in stability at neutral pH, compared to the wild type, and they no longer showed pH dependence of stability. The pK(a)'s of Asp 23 and Glu 9 in these mutant proteins shifted closer to their respective unperturbed values, indicating that the unfavorable electrostatic interactions have been reduced in the mutant proteins. Interestingly, the wild-type and mutant proteins were all stabilized to a similar degree by the addition of 1 M sodium chloride at both neutral and acidic pH, suggesting that the repulsive interactions between the carboxyl groups cannot be effectively shielded by 1 M sodium chloride. These results indicate that repulsive interactions between like charges on the protein surface can destabilize a protein, and protein stability can be significantly improved by relieving these interactions.
一般认为，蛋白质表面的静电相互作用，如离子对，对蛋白质稳定性的贡献很小，尽管它们可能在构象特异性中发挥重要作用。我们发现，人纤连蛋白的第十个III型纤连蛋白结构域（FNfn10）在酸性pH下比中性pH下更稳定，其表观转变中点接近pH 4。对所有天冬氨酸（Asp）和谷氨酸（Glu）残基侧链羧基的pK(a)值进行测定后发现，Asp 23和Glu 9的pK(a)值发生了上移。这些残基与Asp 7在FNfn10表面形成了一个带负电荷的区域，其中Asp 7位于Asp 23和Glu 9之间的中心位置，这表明在中性pH下这些残基之间存在排斥性静电相互作用。分别将Asp 7替换为天冬酰胺（Asn）和赖氨酸（Lys）的突变蛋白D7N和D7K，与野生型相比，在中性pH下稳定性有适度但显著的增加，并且它们不再表现出稳定性的pH依赖性。这些突变蛋白中Asp 23和Glu 9的pK(a)值向各自未受干扰的值移动，表明突变蛋白中不利的静电相互作用已减少。有趣的是，在中性和酸性pH下添加1 M氯化钠后，野生型和突变型蛋白的稳定性都得到了相似程度的提高，这表明羧基之间的排斥相互作用不能被1 M氯化钠有效屏蔽。这些结果表明，蛋白质表面相同电荷之间的排斥相互作用会使蛋白质不稳定，而减轻这些相互作用可以显著提高蛋白质的稳定性。
