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首页> 外文期刊>Biochemistry >Surface Salt Bridges, Double-Mutant Cycles, and Protein Stability: an Experimental and Computational Analysis of the Interaction of the Asp 23 Side Chain with the N-Terminus of the N-Terminal Domain of the Ribosomal Protein L9
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Surface Salt Bridges, Double-Mutant Cycles, and Protein Stability: an Experimental and Computational Analysis of the Interaction of the Asp 23 Side Chain with the N-Terminus of the N-Terminal Domain of the Ribosomal Protein L9

机译：表面盐桥，双突变周期和蛋白质稳定性：核糖体蛋白L9 N末端域的Asp 23侧链与N末端相互作用的实验和计算分析。

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摘要

Experimental and theoretical double-mutant cycles have been used to investigate a salt bridge in the N-terminal domain of the protein L9. Aspartic acid 23 is the only acidic residue involved in a well-defined pairwise interaction, namely, a partially solvent-exposed salt bridge with the protonated N-terminus of the protein. Mutations were studied in which Asp 23 was substituted by alanine, asparagine, and nitrile alanine. Interactions with the N-terminus were probed by comparisons between proteins with a protonated and acetylated N-terminus. The mutants were all folded, and the structures were unchanged from wild type as judged by CD and 2-D NMR. The coupling free energy between the N-terminus and the side chain of Asp 23 measured through double-mutant cycle analysis was favorable and ranged from -0.7 to -1.7 kcal mol~(-1), depending upon the et of mutants used. This relatively large coupling free energy for a surface salt bridge likely arises from geometric factors that reduce the entropy loss associated with salt-bridge formation and from structural relaxation in the mutants. Coupling free energies computed with continuum electrostatic calculations agreed well with the experimental values when full account was taken of all potential interactions particularly those involving Asp 23 and the acetylated N-terminus as well as interactions with solvent. The measured and calculated coupling free energy decreased only slightly when the salt concentration was increased from 100 to 750 mM NaCl. The calculations suggest that the coupling free energy between D23 and the N-terminus measured through the experimental double-mutant cycle analysis is significantly smaller than the actual interaction free energy between the groups in the wild-type structure because of the inapplicability of assumptions frequently used to interpret double-mutant cycles.
机译：实验和理论上的双突变周期已用于研究蛋白质L9 N端结构域中的盐桥。天冬氨酸23是唯一参与明确定义的成对相互作用的酸性残基，即与蛋白质的质子化的N端部分暴露于溶剂的盐桥。研究了其中Asp 23被丙氨酸，天冬酰胺和腈丙氨酸取代的突变。通过比较具有质子化和乙酰化的N末端的蛋白质，可以探测与N末端的相互作用。通过CD和2-D NMR判断，突变体全部折叠，并且结构与野生型没有变化。通过双突变周期分析测得的Asp 23的N末端与侧链之间的偶合自由能是有利的，取决于所用突变体的et，其范围为-0.7至-1.7 kcal mol〜（-1）。用于表面盐桥的相对较大的耦合自由能可能源自降低与盐桥形成相关的熵损失的几何因素以及突变体中的结构弛豫。当充分考虑到所有潜在的相互作用，特别是涉及Asp 23和乙酰化N末端的相互作用以及与溶剂的相互作用时，通过连续静电计算计算出的耦合自由能与实验值非常吻合。当盐浓度从100 mM NaCl增加到750 mM NaCl时，测量和计算的偶合自由能仅略有下降。计算表明，通过实验的双突变周期分析测得的D23和N末端之间的偶合自由能明显小于野生型结构中各组之间的实际相互作用自由能，因为经常使用的假设不适用解释双突变周期。

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