Eriksson A E, Baase W A, Zhang X J, Heinz D W, Blaber M, Baldwin E P, Matthews B W
Institute of Molecular Biology, Howard Hughes Medical Institute, Eugene, OR.
Science. 1992 Jan 10;255(5041):178-83. doi: 10.1126/science.1553543.
Six "cavity-creating" mutants, Leu46----Ala (L46A), L99A, L118A, L121A, L133A, and Phe153----Ala (F153A), were constructed within the hydrophobic core of phage T4 lysozyme. The substitutions decreased the stability of the protein at pH 3.0 by different amounts, ranging from 2.7 kilocalories per mole (kcal mol-1) for L46A and L121A to 5.0 kcal mol-1 for L99A. The double mutant L99A/F153A was also constructed and decreased in stability by 8.3 kcal mol-1. The x-ray structures of all of the variants were determined at high resolution. In every case, removal of the wild-type side chain allowed some of the surrounding atoms to move toward the vacated space but a cavity always remained, which ranged in volume from 24 cubic angstroms (A3) for L46A to 150 A3 for L99A. No solvent molecules were observed in any of these cavities. The destabilization of the mutant Leu----Ala proteins relative to wild type can be approximated by a constant term (approximately 2.0 kcal mol-1) plus a term that increases in proportion to the size of the cavity. The constant term is approximately equal to the transfer free energy of leucine relative to alanine as determined from partitioning between aqueous and organic solvents. The energy term that increases with the size of the cavity can be expressed either in terms of the cavity volume (24 to 33 cal mol-1 A-3) or in terms of the cavity surface area (20 cal mol-1 A-2). The results suggest how to reconcile a number of conflicting reports concerning the strength of the hydrophobic effect in proteins.
在噬菌体T4溶菌酶的疏水核心区域构建了六个“形成空腔”的突变体，分别是Leu46→Ala（L46A）、L99A、L118A、L121A、L133A和Phe153→Ala（F153A）。这些取代使蛋白质在pH 3.0时的稳定性以不同程度降低，范围从L46A和L121A的每摩尔2.7千卡（kcal mol⁻¹）到L99A的5.0 kcal mol⁻¹。还构建了双突变体L99A/F153A，其稳定性降低了8.3 kcal mol⁻¹。所有变体的X射线结构均以高分辨率测定。在每种情况下，去除野生型侧链会使周围一些原子向空出的空间移动，但总是会留下一个空腔，其体积范围从L46A的24立方埃（ų）到L99A的150 ų。在这些空腔中均未观察到溶剂分子。相对于野生型，突变的Leu→Ala蛋白质的去稳定化可以用一个常数项（约2.0 kcal mol⁻¹）加上一个与空腔大小成比例增加的项来近似。该常数项大约等于亮氨酸相对于丙氨酸从水相和有机相之间的分配所确定的转移自由能。随空腔大小增加的能量项可以用空腔体积（24至33 cal mol⁻¹ Å⁻³）或空腔表面积（20 cal mol⁻¹ Å⁻²）来表示。这些结果表明了如何协调关于蛋白质中疏水作用强度的一些相互矛盾的报道。
