Goldenberg D P, Bekeart L S, Laheru D A, Zhou J D
Department of Biology, University of Utah, Salt Lake City 84112.
Biochemistry. 1993 Mar 23;32(11):2835-44. doi: 10.1021/bi00062a015.
The effects of single amino acid replacements on the stability of the 14-38 disulfide bond in the native form of bovine pancreatic trypsin inhibitor (BPTI) were measured. A total of 17 mutant proteins, with substitutions at one of 7 residues located 5-15 A from the disulfide in the native wild-type protein, were examined. The replacements were found to decrease the thermodynamic stability of the disulfide, as measured by exchange with thiol-disulfide reagents, by 0.6-5 kcal/mol, corresponding to a range of nearly 100 mV in redox potentials. The effects of the substitutions on disulfide stability were roughly correlated with the changes in side-chain volume, suggesting that optimal packing is a major factor in determining the stability of the disulfide in the wild-type protein. With only one exception, the substitutions also led to increases, as large as 50-fold, in the rates of disulfide reduction by dithiothreitol. The increased rates of reduction suggest that at least a fraction of the mutational destabilization of the disulfide is due to strain in the native protein that is relieved in the transition state for reduction. The stability of the disulfide in a peptide corresponding to the segments that are linked together by the 14-38 disulfide in native BPTI was found to be about 5 kcal/mol less than that of the disulfide in the intact wild-type protein. Together, the results with the mutant proteins and the peptide indicate that the stability of the disulfide in the native protein depends on both the local environment of the disulfide and on the ability of the rest of the protein to favor a conformation that promotes disulfide formation.
测定了单个氨基酸替换对天然形式牛胰蛋白酶抑制剂（BPTI）中14 - 38二硫键稳定性的影响。总共检测了17种突变蛋白，这些蛋白在天然野生型蛋白中距离二硫键5 - 15埃的7个残基之一处发生了替换。通过与硫醇 - 二硫键试剂交换来测量，发现这些替换会使二硫键的热力学稳定性降低0.6 - 5千卡/摩尔，这对应于氧化还原电位近100毫伏的范围。替换对二硫键稳定性的影响大致与侧链体积的变化相关，这表明最佳堆积是决定野生型蛋白中二硫键稳定性的一个主要因素。除了一个例外，这些替换还导致二硫苏糖醇还原二硫键的速率增加，高达50倍。还原速率的增加表明，二硫键突变失稳至少部分是由于天然蛋白中的应变，这种应变在还原的过渡态中得以缓解。发现与天然BPTI中通过14 - 38二硫键连接在一起的片段相对应的肽中二硫键的稳定性比完整野生型蛋白中的二硫键稳定性低约5千卡/摩尔。综合来看，突变蛋白和肽的结果表明，天然蛋白中二硫键的稳定性既取决于二硫键的局部环境也取决于蛋白其余部分有利于促进二硫键形成的构象的能力。
