NMR Analysis of Pressure-Induced Structural Changes in Prioteins

1. 我们利用实验室开发的一种新型高压核磁共振技术来研究压力对溶液中蛋白质结构的影响。该技术适用于在1到2000巴之间的任何压力下的溶液中的蛋白质，在现代高场核磁共振波谱仪上工作在17.6 T或750 MHz，每秒1小时。我们将该技术应用于许多蛋白质，包括15N均匀标记的BPTI（碱性胰蛋白酶抑制剂），细菌紫质螺旋（1-36），HPr（含组氨酸磷载体蛋白），RalGEF-RBD （Ral鸟嘌呤核苷酸交换因子- ras结合域）。在肽NH基团的15N /1H二维光谱中，压力诱导的1H和15N化学位移变化表明，折叠蛋白的整个主链结构对压力有响应，但在序列上响应是不均匀的。根据压力引起的化学位移，我们得出以下结论：(1)几乎所有酰胺NH基团的氢键距离，无论是羰基还是溶剂水，都在压力作用下缩短；(2)压力诱导的15N位移报告了多肽主链在压力作用下的非均匀构象变化。从压力诱导BPTI的NOE（核过度效应）强度的变化中，我们得出结论，压力诱导蛋白质某些区域的选择性压实，伴随着一些芳香环的翻转运动减慢。最后，从RalGEF-RBD的信号强度测量中，我们发现压力诱导蛋白质优先在1000 bar以上的环区局部展开，然后在2000 bar下展开整个蛋白质。

1. We utilized a novel high pressure NMR technique that was developed in our laboratory to study the effect of pressure on protein structures in solution. The technique works on proteins in solution at any pressure between 1 and 2000 bar on a modern high field NMR spectrometer operating at 17.6 T or 750 MHz for 1H.2. We applied this technique to a number of proteins including 15N -uniformly labeled BPTI (basic pancreatic trypsin inhibitor), helix (1-36) of Bacteriorhodopsin, HPr (histidine-containing phospho-carrier protein), RalGEF-RBD (Ral guanine-nucleotide exchange factor-Ras binding domain). Pressure-induced 1H and 15N chemical shift changes revealed in 15N /1H two-dimensional spectra of peptide NH groups showed that the entire backbone structure of a folded protein responds to pressure, but that the response is non-uniform over the sequence.3. From pressure-induced chemical shifts, we conclude (1) Distances of practically all the hydrogen bonds of amide NH groups, either with carbonyls or solvent water, are shortened by pressure ; (2) Pressure-induced 15N shifts report non-uniform conformational changes in the polypeptide backbone by pressure.4. From pressure-induced changes in NOE (Nuclear Overhauser Effect) intensities for BPTI, we conclude that pressure induces selective compactions of certain regions of the protein, with concomitant slowing down of flip-flop motions of some aromatic rings.5. Finally, from signal intensity measurements in RalGEF-RBD, we found that pressure induces local unfolding of the protein preferentially in loop regions above 1000 bar, followed by unfolding of the entire protein at 2000 bar.