INVESTIGATING RELATIONSHIP OF ISOTOPE SHIFTS & HYDROGEN BONDS IN PROTEINS

同位素对化学位移的影响是众所周知的，而且已经 反映了观测到的原子核的化学环境。 这一效应已被用来研究小分子的氢键。 有机溶剂中的分子。在这个项目中，我们希望调查 同位素位移对探测氢键的作用 水溶液中的蛋白质。最近发生了大量的 对“强”氢键在催化中的可能作用感兴趣。 据信参与这些强氢键的质子 发生强烈下场移动的核磁共振。首字母 实验是为了检测同位素对化学位移的影响 其中一些质子发生了强烈的移位，并建立了 同位素位移、化学位移与官能团的相关性 行为。对于小分子，通常可以测量 氢和氢的主同位素效应对位移的影响 氢气。氘的线宽要大得多。 到它的四极耦合，但对于较短的关联时间 足够锐利，可以准确地确定峰值中心。然而， 对于蛋白质来说，由于它们的关联时间要长得多，所以氢 线条如此之宽，以至于无法被检测到，而对比 必须在质子和氚之间进行。因为涉及到的氢气 不稳定(相对较快地与溶剂交换) 测量必须在氚水中进行(在H或D中约为2%T)。 然后，检测需要抑制来自 水，但这是以与溶剂抑制相同的方式完成的 通常用于检测质子化水中的质子信号。 已经对核糖核酸酶A和胰凝乳酶进行了初步研究。对于RNase 在10 mm浓度下，相当宽的下场移位共振 可以很容易地在氚光谱中检测到。线路13.736， 13.162和12.470 ppm的同位素位移分别为-0.177、-0.116和-0.099 分别为ppm。因此，似乎确实存在着普遍的相关性 化学位移和同位素位移。在凝乳酶中，样品只有 2 mm，抑制的质子共振约为18.5ppm 在氚光谱中看不到酶。S的实验将 努力重复，以优化检测的灵敏度，以及 如果需要，具有更长的获取时间。另一项实验是 计划进行T-15N关联，以检测T和T的同位素效应 和15N在葡萄球菌核酸酶样品中。

Isotope effects on chemical shift are well known, and have been shown to reflect the chemical environment of the observed nucleus. This effect has been exploited to study hydrogen bonding within small molecules in organic solvents. In this project we wish to investigate the utility of the isotope shifts for probing hydrogen bonds within protein in aqueous solution. There has been a great deal of recent interest in the possible role of "strong" hydrogen bonds in catalysis. Protons believed to be involved in these strong hydrogen bonds have NMR resonances that are strongly downfield shifted. Initial experiments are to examine the isotope effect on chemical shift of some of these strongly shifted protons, and establish the degree of correlations between isotope shift, chemical shift and functional behavior. For small molecules it has often been possible to measure the primary isotope effect on shift by comparing hydrogen and deuterium. The linewidths for deuterium are substantially larger due to its quadrupole coupling, but for short correlation times the lines are sharp enough to accurately determine the peak centers. However for proteins, with their much longer correlation times, deuterium lines are so broad that they cannot be detected, and the comparison must be done between proton and tritium. Since the hydrogens involved are labile (exchanging relatively rapidly with solvent) the measurements must be done in tritiated water (ca. 2% T in H or D). Detection then requires suppression of the bulk tritium signal from water, but this is done in the same way that solvent suppression is normally done for detection of proton signals in protonated water. Initial studies have been done on RNase A and chymotrypsin. For RNase at 10 mM concentration the fairly broad, downfield shifted resonances could easily be detected in the tritium spectrum. Lines at 13.736, 13.162 and 12.470 ppm had isotope shifts of -0.177, -0.116 and -0.099 ppm respectively. Thus there does seem to be a general correlation of chemical shift and isotope shift. In chymotrypsin the sample was only 2 mM, and the proton resonance at about 18.5 ppm in the inhibited enzyme was not visible in the tritium spectrum. Thi s experiment will be repeated with effort to optimize the sensitivity of detection, and with longer acquisition time if needed. An additional experiment is scheduled to do T-15N correlations to detect isotope effects on both T and 15N in a sample of Staphylococcus nuclease.