Probing the determinants of disulfide stability in native pancreatic trypsin inhibitor.
Probing the determinants of disulfide stability in native pancreatic trypsin inhibitor.
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探讨天然胰腺胰蛋白酶抑制剂中二硫键稳定性的决定因素。
DOI：
10.1021/bi00062a015
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Zhou,JD
中科院分区：
文献类型：
--
作者：
Goldenberg,DP;Bekeart,LS;Laheru,DA;Zhou,JD
本文测定了单个氨基酸置换对牛胰蛋白酶抑制剂（BPTI）天然形式中14-38二硫键稳定性的影响。共检测了17个突变蛋白，其中在天然野生型蛋白中的二硫化物的5-15 A处的7个残基之一处具有取代。通过与硫醇-二硫化物试剂交换，发现这些取代使二硫化物的热力学稳定性降低0.6-5 kcal/mol，对应于氧化还原电位接近100 mV的范围。取代对二硫键稳定性的影响大致与侧链体积的变化相关，表明最佳包装是决定野生型蛋白中二硫键稳定性的主要因素。只有一个例外，取代也导致增加，高达50倍，在二硫键还原率二硫苏糖醇。还原速率的增加表明，至少有一部分的突变不稳定的二硫化物是由于应变在天然蛋白质中，这是减轻在过渡态还原。天然BPTI中14-38位二硫键连接在一起的肽段中二硫键的稳定性比完整野生型蛋白中二硫键的稳定性低约5 kcal/mol。总之，突变蛋白质和肽的结果表明，天然蛋白质中二硫键的稳定性取决于二硫键的局部环境和蛋白质其余部分有利于促进二硫键形成的构象的能力。早期关于二硫键在蛋白质折叠和稳定性中的作用的讨论集中在二硫键或任何其他交联，会对蛋白质的未折叠状态产生什么影响。简单的聚合物统计学计算表明，交联可以降低未折叠蛋白质的熵，从而增加天然状态的相对稳定性，其量对应于3-5 kcal/mol（Schellman，1955; Flory，1956;波兰& Scheraga，1965;约翰逊等人，1978年）。然而，最近，二硫键稳定折叠构象的能力也取决于二硫键在天然蛋白质中的环境，这一点变得越来越明显。这一结论来自于对天然存在的二硫化物在蛋白质折叠和稳定性中的作用的研究（Creighton，1983; Creighton & Goldenberg，1984; Lin & Kim，1989），以及利用基因工程引入新的二硫化物来稳定蛋白质的尝试[例如，佩里和韦策尔（1984），潘托利亚诺等人（1989），（1987），Mitchinson和威尔斯（1989），以及Matsumura等人，（1989）]。这两种类型的研究表明，二硫化物的贡献可以根据其在天然蛋白质中的位置而变化很大。由于二硫键是可逆的共价键，其形成的热力学和动力学可以通过以下与硫醇-二硫键的交换直接测量
Revised Manuscript Received December 28, 1992 abstract: The effects of single amino acid replacements on the stability of the 14-38 disulfide bond in the native form of bovine pancreatictrypsin 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, correspondingto a range of nearly 100 mV in redox potentials. The effects of the substitutions on disulfide stability were roughly correlated with thechanges 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 proteinthat is relieved in the transition state for reduction. The stability of the disulfide in a peptide corresponding to the segments that are linked togetherby the 14-38 disulfidein 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 indicatethat 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.Early discussions of the roles of disulfide bonds in protein folding and stability focused on the effect that a disulfide, or any other cross-link, would have on the unfolded state of the protein. Simple polymer statistics calculations indicated that cross-links can reduce the entropy of an unfolded protein, and thereby increase the relative stability of the native state, by an amount corresponding to 3-5 kcal/mol (Schellman, 1955; Flory, 1956; Poland & Scheraga, 1965; Johnson et al., 1978). More recently, however, it has becomeapparent that the ability of a disulfide to stabilize the folded conformation also depends on the environment of the disulfide in the native protein. This conclusion has emerged from studies of the roles of naturallyoccurring disulfides in protein folding and stability (Creighton, 1983; Creighton & Goldenberg, 1984; Lin & Kim, 1989) and from attempts to stabilize proteins using genetic engineering to introduce new disulfides [eg, Perry and Wetzel (1984), Pantoliano et al.(1987), Mitchinson and Wells (1989), and Matsumura et al.(1989)]. Both types of study have shown that the contributions of disulfides can vary greatly depending on their location in the native protein. Because disulfides are reversible covalent bonds, the thermodynamics and kinetics of their formation can be measured directly by following exchange with thiol-disulfide
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影响因子：
5.6
作者：
CREIGHTON, TE;GOLDENBERG, DP
通讯作者：
GOLDENBERG, DP
关于硫醇枯草杆菌蛋白酶的硫醇基团的反应性。
DOI：
--
发表时间：
1973
期刊：
European Journal of Biochemistry
影响因子：
--
作者：
L. Polgár;P. Halász;E. Moravcsik
通讯作者：
E. Moravcsik
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影响因子：
5.6
作者：
Y. Goto;K. Hamaguchi
通讯作者：
K. Hamaguchi
胰腺胰蛋白酶抑制剂折叠和展开的能量。
影响因子：
5.6
作者：
T. E. Creighton
通讯作者：
T. E. Creighton
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影响因子：
2.9
作者：
N. Tan;E. Kaiser
通讯作者：
E. Kaiser