Takano K, Yamagata Y, Yutani K
Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan.
Biochemistry. 2001 Apr 17;40(15):4853-8. doi: 10.1021/bi002792f.
It has been generally believed that polar residues are usually located on the surface of protein structures. However, there are many polar groups in the interior of the structures in reality. To evaluate the contribution of such buried polar groups to the conformational stability of a protein, nonpolar to polar mutations (L8T, A9S, A32S, I56T, I59T, I59S, A92S, V93T, A96S, V99T, and V100T) in the interior of a human lysozyme were examined. The thermodynamic parameters for denaturation were determined using a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. If a polar group had a heavy energy cost to be buried, a mutant protein would be remarkably destabilized. However, the stability (Delta G) of the Ala to Ser and Val to Thr mutant human lysozymes was comparable to that of the wild-type protein, suggesting a low-energy penalty of buried polar groups. The structural analysis showed that all polar side chains introduced in the mutant proteins were able to find their hydrogen bond partners, which are ubiquitous in protein structures. The empirical structure-based calculation of stability change (Delta Delta G) [Takano et al. (1999) Biochemistry 38, 12698--12708] revealed that the mutant proteins decreased the hydrophobic effect contributing to the stability (Delta G(HP)), but this destabilization was recovered by the hydrogen bonds newly introduced. The present study shows the favorable contribution of polar groups with hydrogen bonds in the interior of protein molecules to the conformational stability.
人们普遍认为极性残基通常位于蛋白质结构的表面。然而，实际上在结构内部存在许多极性基团。为了评估这种埋藏的极性基团对蛋白质构象稳定性的贡献，研究了人溶菌酶内部的非极性到极性突变（L8T、A9S、A32S、I56T、I59T、I59S、A92S、V93T、A96S、V99T和V100T）。使用差示扫描量热仪测定变性的热力学参数，并通过X射线晶体学分析晶体结构。如果一个极性基团被埋藏需要付出巨大的能量代价，那么突变蛋白将明显不稳定。然而，丙氨酸到丝氨酸以及缬氨酸到苏氨酸突变的人溶菌酶的稳定性（ΔG）与野生型蛋白相当，这表明埋藏的极性基团具有较低的能量代价。结构分析表明，突变蛋白中引入的所有极性侧链都能够找到它们在蛋白质结构中普遍存在的氢键伙伴。基于经验结构的稳定性变化计算（ΔΔG）[Takano等人（1999年）《生物化学》38卷，12698 - 12708页]表明，突变蛋白降低了对稳定性有贡献的疏水效应（ΔG(HP)），但这种不稳定通过新引入的氢键得以恢复。本研究表明蛋白质分子内部具有氢键的极性基团对构象稳定性有有利贡献。
