NMR Analysis of Pressure-Induced Structural Changes in Prioteins

1.我们利用一种新的高压核磁共振技术，在我们的实验室开发的研究压力对蛋白质结构的影响在溶液中。该技术适用于溶液中的蛋白质，在1至2000 bar之间的任何压力下，在17.6 T或750 MHz的现代高场NMR光谱仪上进行1H.2。我们将该技术应用于许多蛋白质，包括15 N-均匀标记的BPTI（碱性胰蛋白酶抑制剂）、细菌视紫红质的螺旋（1-36）、HPr（含组氨酸的磷酸载体蛋白）、RalGEF-RBD（Ral鸟嘌呤-核苷酸交换因子-Ras结合结构域）。压力诱导的1H和15 N化学位移变化揭示了肽NH基团的15 N/1H二维光谱，表明折叠蛋白质的整个骨架结构响应于压力，但响应在序列上是不均匀的.从压力诱导的化学位移，我们得出结论：（1）几乎所有的酰胺NH基团的氢键的距离，无论是与羰基或溶剂水，被压力缩短;（2）压力诱导的15 N位移报告非均匀构象变化的多肽主链的压力.从压力诱导的NOE（核奥弗豪泽效应）强度的变化中，我们得出结论，压力诱导蛋白质的某些区域的选择性压缩，伴随着一些芳香环的翻转运动的减慢。最后，从RalGEF-RBD中的信号强度测量，我们发现压力优先在1000 bar以上的环区域诱导蛋白质的局部解折叠，然后在2000 bar下诱导整个蛋白质的解折叠。

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.