Kellis J T, Nyberg K, Fersht A R
Department of Chemistry, University of Cambridge, U.K.
Biochemistry. 1989 May 30;28(11):4914-22. doi: 10.1021/bi00437a058.
The energetics of complementary packing of nonpolar side chains in the hydrophobic core of a protein were analyzed by protein engineering experiments. We have made the mutations Ile----Val, Ile----Ala, and Leu----Ala in a region of the small bacterial ribonuclease barnase where the major alpha-helix packs onto the central beta-sheet. The destabilization resulting from the creation of cavities was determined by measuring the decrease in free energy of folding from reversible denaturation induced by urea, guanidinium chloride, or heat. The different methods give consistent and reproducible results. The loss in free energy of folding for the mutant proteins is 1.0-1.6 kcal/mol per methylene group removed. This exceeds by severalfold the values obtained from model experiments of the partitioning of relevant side chains between aqueous and nonpolar solvents. Much of this discrepancy arises because two surfaces are buried when a protein folds--both the amino acid side chain in question and the portions of the protein into which it packs. These experiments directly demonstrate that the interior packing of a protein is crucial in stabilizing its three-dimensional structure: the conversion of leucine or isoleucine to alanine in the hydrophobic core loses half the net free energy of folding of barnase with a concomitant decrease in yield of the expressed recombinant protein.
通过蛋白质工程实验分析了蛋白质疏水核心中非极性侧链互补堆积的能量学。我们在小细菌核糖核酸酶巴那斯酶的一个区域进行了Ile→Val、Ile→Ala和Leu→Ala的突变，该区域主要的α螺旋堆积在中央β折叠片上。通过测量由尿素、氯化胍或热诱导的可逆变性导致的折叠自由能的降低，确定了由形成空洞引起的去稳定化。不同的方法给出了一致且可重复的结果。突变蛋白折叠自由能的损失为每去除一个亚甲基1.0 - 1.6千卡/摩尔。这比从相关侧链在水相和非极性溶剂之间分配的模型实验获得的值高出几倍。这种差异很大程度上是因为蛋白质折叠时两个表面被掩埋——所讨论的氨基酸侧链以及它所堆积的蛋白质部分。这些实验直接证明了蛋白质的内部堆积对于稳定其三维结构至关重要：在疏水核心中亮氨酸或异亮氨酸向丙氨酸的转变使巴那斯酶折叠的净自由能损失一半，同时表达的重组蛋白产量降低。
