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， 这反映了观察到的原子核的化学环境。 这种效应已被用来研究小分子内的氢键。 有机溶剂中的分子。 在这个项目中，我们希望调查 利用同位素位移来探测 水溶液中的蛋白质。 最近有很多 在催化作用中的“强”氢键的可能作用的兴趣。 据信与这些强氢键有关的质子 强烈低场位移的NMR共振。 初始 实验是为了研究同位素对化学位移的影响， 其中一些强烈移动的质子，并建立了 同位素位移、化学位移和泛函之间的相关性 行为 对于小分子，通常可以测量 通过比较氢和氢同位素， 氘 氘的线宽基本上更大， 它的四极耦合，但短相关时间的线 足够精确地确定峰值中心。 然而 对于蛋白质，由于它们的相关时间要长得多，氘 标题：研究同位素位移与 蛋白质中的氢键（续）如此广泛，以至于它们不能被 检测到，并且必须在质子和氚之间进行比较。 由于所涉及的氢是不稳定的（交换相对较快 使用溶剂）必须在氚化水（ca. H或D中的2% T）。 然后检测需要抑制大部分 氚信号，但这是以同样的方式完成的， 通常进行溶剂抑制以检测质子信号 在质子化的水中。 已经对RNase A进行了初步研究， 糜蛋白酶 对于10 mM浓度的RNA酶， 低场位移共振可以很容易地检测到的氚 江西篇章 13.736、13.162和12.470 ppm处的谱线发生了同位素位移 分别为-0.177、-0.116和-0.099 ppm。 因此， 是化学位移和同位素位移的一般关系。 在 胰凝乳蛋白酶的样品是只有2 mM，和质子共振在 约18.5ppm的被抑制的酶在氚中不可见 江西篇章 将重复此实验，以优化 检测的灵敏度，并且如果 needed. T-15 N还计划进行一次额外的实验 检测样品中T和15 N的同位素效应的相关性 葡萄球菌核酸酶

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 TITLE: Investigating the Relationship of Isotope Shifts and Hydrogen Bonds in Proteins (Continued) 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. This 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.