Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN, 47907-2084, USA.
J Biomol NMR. 2010 Mar;46(3):227-44. doi: 10.1007/s10858-010-9398-8. Epub 2010 Feb 27.
Amide solvent exchange rates are regarded as a valuable source of information on structure/dynamics of unfolded (disordered) proteins. Proton-based saturation transfer experiments, normally used to measure solvent exchange, are known to meet some serious difficulties. The problems mainly arise from the need to (1) manipulate water magnetization and (2) discriminate between multiple magnetization transfer pathways that occur within the proton pool. Some of these issues are specific to unfolded proteins. For example, the compensation scheme used to cancel the Overhauser effect in the popular CLEANEX experiment is not designed for use with unfolded proteins. In this report we describe an alternative experimental strategy, where amide (15)N is used as a probe of solvent exchange. The experiment is performed in 50% H(2)O-50% D(2)O solvent and is based on the (HACACO)NH pulse sequence. The resulting spectral map is fully equivalent to the conventional HSQC. To fulfill its purpose, the experiment monitors the conversion of deuterated species, (15)N(D), into protonated species, (15)N(H), as effected by the solvent exchange. Conceptually, this experiment is similar to EXSY which prompted the name of (15)N(H/D)-SOLEXSY (SOLvent EXchange SpectroscopY). Of note, our experimental scheme, which relies on nitrogen rather than proton to monitor solvent exchange, is free of the complications described above. The developed pulse sequence was used to measure solvent exchange rates in the chemically denatured state of the drkN SH3 domain. The results were found to correlate well with the CLEANEX-PM data, r = 0.97, thus providing a measure of validation for both techniques. When the experimentally measured exchange rates are converted into protection factors, most of the values fall in the range 0.5-2, consistent with random-coil behavior. However, elevated values, ca. 5, are obtained for residues R38 and A39, as well as the side-chain indole of W36. This is surprising, given that high protection factors imply hydrogen bonding or hydrophobic burial not expected to occur in a chemically denatured state of a protein. We, therefore, hypothesized that elevated protection factors are an artefact arising from the calculation of the reference (random-coil) exchange rates. To confirm this hypothesis, we prepared samples of several short peptides derived from the sequence of the drkN SH3 domain; these samples were used to directly measure the reference exchange rates. The revised protection factors obtained in this manner proved to be close to 1.0. These results also have implications for the more compact unfolded state of drkN SH3, which appears to be fully permeable to water as well, with no manifestations of hydrophobic burial.
酰胺溶剂交换率被认为是研究无规（无序）蛋白质结构/动力学的有价值的信息来源。通常用于测量溶剂交换的质子饱和转移实验已知存在一些严重的困难。这些问题主要源于需要 (1) 操纵水磁化和 (2) 区分质子池内发生的多种磁化转移途径。其中一些问题是针对无规蛋白质的。例如，用于消除流行的 CLEANEX 实验中 Overhauser 效应的补偿方案并非专为无规蛋白质设计。在本报告中，我们描述了一种替代实验策略，其中酰胺 (15)N 用作溶剂交换的探针。该实验在 50% H2O-50% D2O 溶剂中进行，并基于 (HACACO)NH 脉冲序列。所得的光谱图与常规 HSQC 完全等效。为了实现其目的，该实验监测由溶剂交换引起的氘代物种 (15)N(D)向质子化物种 (15)N(H)的转化。从概念上讲，该实验类似于 EXSY，这促使该实验被命名为 (15)N(H/D)-SOLEXSY（SOLvent EXchange SpectroscopY）。值得注意的是，我们的实验方案依赖于氮而不是质子来监测溶剂交换，因此没有上述复杂情况。所开发的脉冲序列用于测量 drkN SH3 结构域化学变性状态下的溶剂交换速率。结果与 CLEANEX-PM 数据相关性良好，r = 0.97，因此为两种技术提供了验证。当实验测量的交换率转换为保护因子时，大多数值落在 0.5-2 的范围内，与无规卷曲行为一致。然而，对于残基 R38 和 A39 以及侧链吲哚 W36，获得的数值约为 5，这令人惊讶，因为高保护因子意味着氢键或疏水掩埋不应发生在蛋白质的化学变性状态下。因此，我们假设高保护因子是由于计算参考（无规卷曲）交换率而产生的伪影。为了证实这一假设，我们制备了源自 drkN SH3 结构域序列的几个短肽的样品；这些样品用于直接测量参考交换率。以这种方式获得的修正保护因子接近于 1.0。这些结果也对 drkN SH3 更紧凑的无规状态具有影响，该状态似乎也完全可渗透水，没有疏水掩埋的迹象。
