EFFECT OF CAVITY-CREATING MUTATIONS IN THE HYDROPHOBIC CORE OF CHYMOTRYPSIN INHIBITOR-2
EFFECT OF CAVITY-CREATING MUTATIONS IN THE HYDROPHOBIC CORE OF CHYMOTRYPSIN INHIBITOR-2
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DOI：
10.1021/bi00093a001
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发表时间：
1993-10-26
期刊：
影响因子：
2.9
通讯作者：
FERSHT, AR
中科院分区：
文献类型：
--
作者：
JACKSON, SE;MORACCI, M;FERSHT, AR
一种截短形式的凝乳酶抑制因子2(CI2)核心中的疏水残基发生了突变，以测量它们对蛋白质稳定性的贡献。用氯化胍诱导的变性和差示扫描量热法测定野生型和突变体的去折叠自由能。这两种方法给出了自由能随突变的变化在1%或2%范围内的结果。Ile-->；Val突变的去折叠自由能(+/-标准差)的平均变化为1.2+/-0.1kcal mol-1，Val-->；Ala突变的平均展开自由能变化为3.4+/-1.5kcal mol-1，Ile-->；Ala或Leu-->；Ala突变的平均展开自由能变化为3.6+/-0.6kcal-1。这给出了删除1.3+/-0.5千卡摩尔-1的一个亚甲基的展开自由能的平均变化。野生型和突变型DELTADELTAG(U-F)之间展开自由能的变化与蛋白质中突变残基的环境有两个显著的相关性。第一个是DELTADELTAG(U-F)与野生型和突变体侧链溶剂可及区之间的差异(相关系数=0.81，10分)。DELTADELTAG(U-F)与N之间存在较好的相关性(相关系数=0.84，10分)。N是指被删除的疏水基团半径6角内的甲基/亚甲基基团的数量。后一种关联与之前发现的Barnase非常相似，这表明这种关系是普遍的，并适用于其他球状蛋白的疏水核心。C12和Barnase的组合数据与N(相关系数=0.87，30点)的相关性明显好于与溶剂可及表面积的变化(相关系数=0.82，30点)。这表明，特定残基周围的堆积密度对于确定残基对蛋白质稳定性的贡献是重要的。在一个案例中，Ile-->；Val76，一种缺失埋藏侧链的C(Delta1)甲基的突变，获得了一个令人惊讶的结果。该突变体的稳定性比野生型高0.2+/-0.1kcal·mol-1。我们对该突变体的晶体结构进行了求解和分析，发现核心中存在侧链的微小运动，其中最大的0.7埃是发生突变的侧链的运动。这些微小的运动在一定程度上弥补了突变造成的空洞。
Hydrophobic residues in the core of a truncated form of chymotrypsin inhibitor 2 (CI2) have been mutated in order to measure their contribution to the stability of the protein. The free energy of unfolding of wild-type and mutants was measured by both guanidinium chloride-induced denaturation and differential scanning calorimetry. The two methods give results for the changes in free energy on mutation that agree to within 1% or 2%. The average change in the free energy of unfolding (+/- standard deviation) for an Ile --> Val mutation is 1.2 +/- 0.1 kcal mol-1, for a Val --> Ala mutation 3.4 +/- 1.5 kcal mol-1, and for either an Ile --> Ala or a Leu --> Ala mutation 3.6 +/- 0.6 kcal mol-1. This gives an average change in the free energy of unfolding for deleting one methylene group of 1.3 +/- 0.5 kcal mol-1. Two significant correlations were found between the change in the free energy of unfolding between wild-type and mutant, DELTADELTAG(U-F), and the environment of the mutated residue in the protein. The first is between DELTADELTAG(U-F) and the difference in side-chain solvent-accessible area buried between wild-type and mutant (correlation coefficient = 0.81, 10 points). The second and slightly better correlation was found between DELTADELTAG(U-F) and N, the number of methyl/methylene groups within a 6-angstrom radius of the hydrophobic group deleted (correlation coefficient = 0.84, 10 points). The latter correlation is very similar to that found previously for barnase, suggesting that this relationship is general and applies to the hydrophobic cores of other globular proteins. The combined data for C12 and barnase clearly show a better correlation with N (correlation coefficient = 0.87, 30 points) than with the change in' the solvent-accessible surface area (correlation coefficient = 0.82, 30 points). This indicates that the packing density around a particular residue is important in determining the contribution the residue makes to protein stability. In one case, Ile --> Val76, a mutation which deletes the C(delta1) methyl group of a buried side chain, a surprising result was obtained. This mutant was found to be more stable than wild-type by 0.2 +/- 0.1 kcal mol-1. We have solved and analyzed the crystal structure of this mutant and find that there are small movements of side chains in the core, the largest of which, 0.7 angstrom, is a movement of the side chain that has been mutated. These small movements in some part compensate for the cavity created by the mutation.