Additivity of differential conformational dynamics in hyperthermophile/mesophile rubredoxin chimeras as monitored by hydrogen exchange.
Additivity of differential conformational dynamics in hyperthermophile/mesophile rubredoxin chimeras as monitored by hydrogen exchange.
复制标题
通过氢交换监测超嗜热/嗜温红氧还蛋白嵌合体中差异构象动力学的加和性。
DOI：
10.1002/cbic.200600276
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Hernandez,Griselda
中科院分区：
文献类型：
--
作者：
LeMaster,DavidM;Hernandez,Griselda
瞬时构象动力学一直被认为在蛋白质的生物学功能中起着至关重要的作用。最近，更直接的证据表明，构象波动在催化作用中的作用已经被发现的酶进行协调过渡的活性位点内发生的时间框架内的底物营业额。[1-4]破坏这些集体活性位点转换的突变导致催化作用降低。[5，6]监测远离突变位点的残基的内部移动性可以深入了解构象动力学如何通过蛋白质结构传播。然而，在这样的研究中，远端位点动力学的实质性变化的证据通常来自于产生全局稳定性显著降低的突变。[7-10]由这种不稳定突变引起的天然样相互作用的破坏使在反映亲本蛋白质结构的运动方面对差异构象动力学的解释复杂化。详细的突变分析的构象动力学必须面对的问题是熟悉的定量从经典的蛋白质热力学稳定性分析：由一个给定的取代所产生的不稳定（或改变流动性）的程度往往不是一个令人满意的措施提供的贡献由原来的残基的全局稳定性（或构象动力学）的天然蛋白质。一对结构上同源的蛋白质之间的一个或多个残基的交换，只有当互补的杂交序列产生相对于亲本蛋白质的稳定性的严格相反的变化时，才能被指定为差异热力学稳定性的特定部分。类似地，可以询问是否可以分配一对同源蛋白质的不同残基，使得所得互补杂合蛋白质对的差异构象动力学表现出相似的加和性特性。对于天然进化的差异蛋白质稳定性的分析[11-13]和热稳定性的系统工程[14-17]，最常见的成功是在每个单个残基的交换导致稳定性增加的情况下获得的。在最直接的情况下，例如来自嗜热和嗜温生物体的冷休克蛋白[18]和细胞色素c551 [19，20]的同源对，由一小组残基引起的稳定性变化是独立加和的，并解释了亲本蛋白质的完全差异稳定性。然而，当非保守残基之间存在强相互作用时，就不能再预期这种简单的稳定性加和性。在这种情况下，相互作用的残基可能需要作为簇交换以获得稳定性的净增加。类似的逻辑适用于协同构象转变中隐含的相互作用。一般来说，波动残基簇内的单点取代不能保持微分亲样动力学行为。广泛使用的点突变双突变循环分析[21]证明需要比较互补突变以获得稳定性数据的定量解释。虽然嵌合蛋白构建通常用于分析更广泛的序列变化的影响，但很少分析嵌合蛋白及其互补序列。部分地，这反映了常规基因改组技术[22]生成两个互补序列的可能性较低。一个值得注意的例外是核糖核酸酶H，其中一对互补的...
Transient conformational dynamics have long been inferred to play a critical role in the biological functions of proteins. Recently, more direct evidence for the role of conformational fluctuations in catalysis has been found for enzymes which undergo concerted transitions in the active site that occur within the timeframe of substrate turnover.[1–4] Mutations which disrupt these collective active site transitions result in reduced catalysis.[5, 6] Monitoring the internal mobility of residues that are distant from the mutation site can provide insight into how conformational dynamics propagate through the protein structure. However, in such studies the evidence of substantial changes in dynamics at distal sites have generally arisen from mutations that produce a significant decrease in global stability.[7–10] The disruption of native-like interactions that result from such destabilizing mutations complicate the interpretation of the differential conformational dynamics in terms of motions that reflect the parental protein structure. Detailed mutational analysis of conformational dynamics must confront the problem of quantitation that is familiar from the classical analysis of protein thermodynamic stability: the degree of destabilization (or altered mobility) arising from a given substitution is often not a satisfactory measure of the contribution provided by the original residue to the global stability (or conformational dynamics) of the native protein. The exchange of one or more residues between a pair of structurally homologous proteins can be assigned a specific fraction of the differential thermodynamic stability only if the complementary hybrid sequence yields a strictly opposite change in stability relative to that of the parental proteins. In analogy, it may be asked whether the differing residues for a pair of homologous proteins can be partitioned such that the differential conformational dynamics of the resultant pair of complementary hybrid proteins exhibit a similar property of additivity. For both the analysis of naturally evolved differential protein stability [11–13] and the systematic engineering of thermostability,[14–17] success has most often been obtained for cases in which the exchange of each individual residue results in an increase in stability. In the most straightforward cases, such as the homologous pairs of cold shock protein [18] and cytochrome c551 [19, 20] from thermophilic and mesophilic organisms, the changes in stability arising from a small set of residues are independently additive and account for the full differential stability of the parental proteins. However, when there are strong interactions between the nonconserved residues, this simple additivity in stability can no longer be anticipated. In such cases, the interacting residues may need to be exchanged as a cluster to gain a net increase in stability. A similar logic applies to the mutual interactions implicit in concerted conformational transitions. Single point substitutions within the cluster of fluctuating residues can not, in general, be expected to preserve the differential parental-like dynamical behavior. The widely utilized double mutant cycle analysis of point mutations [21] demonstrates the need to compare the complementary mutation to obtain quantitative interpretation of stability data. Although chimeric protein constructions have been commonly used to analyze the effects of more extensive sequence changes, far less often have both the chimeric protein and its complementary sequence been analyzed. In part, this reflects the low probability for conventional gene-shuffling techniques [22] to generate both of the complementary sequences. A notable exception is that of ribonuclease H for which a pair of complementary …