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Sensitivity-enhanced three-dimensional and carbon-detected two-dimensional NMR of proteins using hyperpolarized water.
Journal of Biomolecular NMR ( IF 2.4 ) Pub Date : 2020-02-10 , DOI: 10.1007/s10858-020-00301-5
Gregory L Olsen 1, 2 , Or Szekely 2 , Borja Mateos 3 , Pavel Kadeřávek 4, 5 , Fabien Ferrage 5 , Robert Konrat 3 , Roberta Pierattelli 6 , Isabella C Felli 6 , Geoffrey Bodenhausen 5 , Dennis Kurzbach 1, 5 , Lucio Frydman 2
Affiliation  

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarization-selective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

中文翻译:

使用超极化水对蛋白质进行灵敏度增强的三维和碳检测的二维NMR。

通过将HDO用作载体将超极化作用引入折叠或固有无序的蛋白质中,可以实现蛋白质NMR信号增强多达两个数量级。在这种方法中,将由溶解动态核极化(D-DNP)产生的超极化HDO与蛋白质溶液混合,并在高场NMR光谱仪中等待,然后酰胺质子交换和核Overhauser效应(NOE)将超极化转移到蛋白质上,能够获取信号增强的高分辨率频谱。迄今为止,该策略的使用仅限于1D和1H-15N 2D相关实验。在这里,我们介绍2D 13C检测到的D-DNP,以减少交换诱导的加宽和其他可能对质子检测到的D-DNP实验产生不利影响的弛豫惩罚。我们还介绍了超极化3D光谱学,为较大的蛋白质和IDP进行D-DNP研究提供了可能性,其中光谱拥挤可能会阻碍分配和残基特异性研究。获得的信号增强尤其取决于所观察到的残基的化学和磁性交换速率,因此导致非均匀的“超极化选择性”信号增强。但是,由此产生的光谱稀疏性使得在超过一分钟的采集时间就可以解析和监控200多个残基的IDP中的单个氨基酸。我们将建议的实验应用于两个模型系统:紧密折叠的蛋白泛素和固有无序蛋白(IDP)骨桥蛋白(OPN)。频谱拥挤可能会阻碍分配和残基特异性研究。获得的信号增强尤其取决于观察到的残基的化学和磁性交换速率,因此导致非均匀的“超极化选择性”信号增强。但是,由此产生的光谱稀疏性使得在超过一分钟的采集时间就可以解析和监控200多个残基的IDP中的单个氨基酸。我们将建议的实验应用于两个模型系统:紧密折叠的蛋白泛素和固有无序蛋白(IDP)骨桥蛋白(OPN)。频谱拥挤可能会阻碍分配和残基特异性研究。获得的信号增强尤其取决于观察到的残基的化学和磁性交换速率,因此导致非均匀的“超极化选择性”信号增强。但是,由此产生的光谱稀疏性使得在超过一分钟的采集时间就可以解析和监控200多个残基的IDP中的单个氨基酸。我们将建议的实验应用于两个模型系统:紧密折叠的蛋白泛素和固有无序蛋白(IDP)骨桥蛋白(OPN)。因此导致非均匀的“超极化选择性”信号增强。但是,由此产生的光谱稀疏性使得在超过一分钟的采集时间就可以解析和监控200多个残基的IDP中的单个氨基酸。我们将建议的实验应用于两个模型系统:紧密折叠的蛋白泛素和固有无序蛋白(IDP)骨桥蛋白(OPN)。因此导致非均匀的“超极化选择性”信号增强。但是,由此产生的光谱稀疏性使得在超过一分钟的采集时间就可以解析和监控200多个残基的IDP中的单个氨基酸。我们将建议的实验应用于两个模型系统:紧密折叠的蛋白泛素和固有无序蛋白(IDP)骨桥蛋白(OPN)。
更新日期:2020-04-21
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