通过二维HMQC NMR评估折叠和未折叠蛋白质中超极化水赋予的位点特异性增强
Assessing site-specific enhancements imparted by hyperpolarized water in folded and unfolded proteins by 2D HMQC NMR
摘要：
超极化水可以是蛋白质NMR中的有价值的辅助，导致酰胺基团1H极化比它们的热对应物大几个数量级。合适的程序可以利用这一点来提供具有良好分辨率和增强灵敏度的2D 1H-15 N相关性。这些增强取决于酰胺和水之间的交换率，从而产生关于溶剂可及性的诊断信息。本研究将这种HyperW方法应用于表现出各种交换行为的四种蛋白质：PhoA 4，一种未折叠的122个残基片段; barstar，一种完全折叠的核糖核酸酶抑制剂; R17，一种13.3 kDa的系统，在缓慢的相互转换下具有折叠和未折叠的形式;和drkN SH 3，一种蛋白质结构域，其折叠和未折叠的形式迅速交换，并具有温度依赖性的群体比例。对于PhoA 4，HyperW的灵敏度增强大于300倍，正如未折叠蛋白质序列所预期的那样。尽管完全折叠的芽孢杆菌芽孢杆菌R17显示出其展开形式的100倍增强的预期叠加，与更温和的折叠对应物共存。然而，出乎意料的是drkN SH 3的行为，HyperW增强了未折叠但更确定的折叠蛋白质位点。这些优惠的增强反复和可重复地观察到的一些解释，包括水，未折叠和折叠状态之间的三个站点交换磁化转移;交叉相关的超极化结构水和不稳定的侧链质子的弛豫过程;和更快的溶剂交换速率表征某些折叠的网站超过其未折叠的对应物的可能性被认为是占他们。
英文摘要：
Hyperpolarized water can be a valuable aid in protein NMR, leading to amide group 1H polarizations that are orders of magnitude larger than their thermal counterparts. Suitable procedures can exploit this to deliver 2D 1H-15N correlations with good resolution and enhanced sensitivity. These enhancements depend on the exchange rates between the amides and the water, thereby yielding diagnostic information about solvent accessibility. This study applied this HyperW method to four proteins exhibiting a gamut of exchange behaviors: PhoA4, an unfolded 122-residue fragment; barstar, a fully folded ribonuclease inhibitor; R17, a 13.3 kDa system possessing folded and unfolded forms under slow interconversion; and drkN SH3, a protein domain whose folded and unfolded forms interchange rapidly and with temperature-dependent population ratios. For PhoA4 HyperW sensitivity enhancements were larger than 300-fold, as expected for an unfolded protein sequence. Though fully folded barstar also exhibited substantial enhancements; these, however, were not uniform, and according to CLEANEX experiments reflected the solvent-exposed residues. R17 showed the expected superposition of 100-fold enhancements for its unfolded form, coexisting with more modest folded counterparts. Unexpected, however, was the behavior of drkN SH3, for which HyperW enhanced the unfolded but even more certain folded protein sites. These preferential enhancements were repeatedly and reproducibly observed; a number of explanations including three-site exchange magnetization transfers between water, unfolded and folded states; cross-correlated relaxation processes from hyperpolarized structural waters and labile sidechain protons; and the possibility that faster solvent exchange rates characterize certain folded sites over their unfolded counterparts are considered to account for them.
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