Mutational analysis demonstrates that specific electrostatic interactions can play a key role in the denatured state ensemble of proteins
Mutational analysis demonstrates that specific electrostatic interactions can play a key role in the denatured state ensemble of proteins
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DOI：
10.1016/j.jmb.2005.08.019
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发表时间：
2005-10-14
影响因子：
5.6
通讯作者：
Raleigh, DP
中科院分区：
文献类型：
--
作者：
Cho, JH;Raleigh, DP
变性态系综的性质几十年来一直存在争议，这在很大程度上是由于难以表征变性态相互作用的结构和能量学。有越来越多的证据表明，在一些蛋白质的变性状态下，相对非特异性的疏水性聚集，但其他类型的相互作用的特点要少得多。在这里，我们报告了高度特异性的静电相互作用的表征在一个小的α-β蛋白，核糖体蛋白L9（NTL 9）的N-末端结构域的变性状态。已显示Lys 12突变为Met通过破坏变性状态相互作用显著增加NTL 9的稳定性。在这里，我们描述了13个突变体的pH依赖性的稳定性的分析，旨在探测的性质的Lys 12变性状态的相互作用。Lys 12位于蛋白质的富含赖氨酸的区域，但对一组Lys至Met突变体的分析表明，它在变性状态下起着独特的作用。NTL 9中所有酸性残基的突变体的分析表明，Lys 12在变性状态系综中与Asp 8形成特定的非天然静电相互作用。因此，NTL 9的变性状态系综中的电荷-电荷相互作用的分布似乎被少数关键相互作用所偏置，并且与无规卷曲中的预期非常不同。我们建议，这些相互作用不是由本地序列效应编码，而是反映了更远的序列中残基之间的相互作用。这些结果表明，静电以及疏水相互作用可以发挥重要作用的变性状态合奏。(c)2005爱思唯尔有限公司保留所有权利。
The nature of the denatured state ensemble has been controversial for decades owing, in large part, to the difficulty in characterizing the structure and energetics of denatured state interactions. There is increasing evidence for relatively non-specific hydrophobic clustering in the denatured states of some proteins but other types of interactions are much less well characterized. Here, we report the characterization of highly specific electrostatic interactions in the denatured state of a small alpha-beta protein, the N-terminal domain of the ribosomal protein L9 (NTL9). Mutation of Lys12 to Met has been shown to increase the stability of NTL9 significantly through the disruption of denatured state interactions. Here, we describe the analysis of the pH-dependent stability of 13 mutants designed to probe the nature of the Lys12 denatured state interaction. Lys12 is located in a lysine-rich region of the protein but analysis of a set of Lys to Met mutants shows that it plays a unique role in the denatured state. Analysis of mutants of all of the acidic residues in NTL9 shows that Lys12 forms a specific non-native electrostatic interaction with Asp8 in the denatured state ensemble. Thus the distribution of charge-charge interactions in the denatured state ensemble of NTL9 appears to be biased by few key interactions and is very different from that expected in a random coil. We propose that these interactions are not encoded by local sequence effects but rather reflect interactions among residues more distant in sequence. These results demonstrate that electrostatic as well as hydrophobic interactions can play an important role in the denatured state ensemble. (c) 2005 Elsevier Ltd. All rights reserved.