Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
J Mol Biol. 2011 Jan 14;405(2):361-77. doi: 10.1016/j.jmb.2010.10.001. Epub 2010 Nov 6.
The side chains of Lys66, Asp66, and Glu66 in staphylococcal nuclease are fully buried and surrounded mainly by hydrophobic matter, except for internal water molecules associated with carboxylic oxygen atoms. These ionizable side chains titrate with pK(a) values of 5.7, 8.8, and 8.9, respectively. To reproduce these pK(a) values with continuum electrostatics calculations, we treated the protein with high dielectric constants. We have examined the structural origins of these high apparent dielectric constants by using NMR spectroscopy to characterize the structural response to the ionization of these internal side chains. Substitution of Val66 with Lys66 and Asp66 led to increased conformational fluctuations of the microenvironments surrounding these groups, even under pH conditions where Lys66 and Asp66 are neutral. When Lys66, Asp66, and Glu66 are charged, the proteins remain almost fully folded, but resonances for a few backbone amides adjacent to the internal ionizable residues are broadened. This suggests that the ionization of the internal groups promotes a local increase in dynamics on the intermediate timescale, consistent with either partial unfolding or increased backbone fluctuations of helix 1 near residue 66, or, less likely, with increased fluctuations of the charged side chains at position 66. These experiments confirm that the high apparent dielectric constants reported by internal Lys66, Asp66, and Glu66 reflect localized changes in conformational fluctuations without incurring detectable global structural reorganization. To improve structure-based pK(a) calculations in proteins, we will need to learn how to treat this coupling between ionization of internal groups and local changes in conformational fluctuations explicitly.
葡萄球菌核酸酶中 Lys66、Asp66 和 Glu66 的侧链完全被埋藏并主要被疏水物质包围，只有与羧酸氧原子相连的内部水分子除外。这些可离子化的侧链的滴定 pK(a) 值分别为 5.7、8.8 和 8.9。为了用连续静电计算重现这些 pK(a) 值，我们用高介电常数处理了蛋白质。我们通过使用 NMR 光谱来表征这些内部侧链的电离对结构的响应，从而研究了这些高表观介电常数的结构起源。用 Lys66 和 Asp66 替代 Val66 导致这些基团周围的微环境的构象波动增加，即使在 Lys66 和 Asp66 呈中性的 pH 条件下也是如此。当 Lys66、Asp66 和 Glu66 带电荷时，蛋白质仍然几乎完全折叠，但与内部可离子化残基相邻的几个骨架酰胺的共振变宽。这表明内部基团的电离促进了中间时间尺度上局部动力学的增加，这与残基 66 附近的螺旋 1 的局部展开或骨架波动增加一致，或者不太可能与位置 66 的带电侧链的波动增加一致。这些实验证实，内部 Lys66、Asp66 和 Glu66 报告的高表观介电常数反映了构象波动的局部变化，而不会引起可检测的全局结构重排。为了改进基于结构的 pK(a) 计算在蛋白质中，我们将需要学习如何明确地处理内部基团的电离和局部构象波动变化之间的这种耦合。
