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 线是如此之宽，以至于它们无法被检测到， 必须在质子和氚之间进行。 因为氢原子 是不稳定的（与溶剂相对快速地交换）， 测量必须在氚化水（ca. H或D中的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进行的。 通常用于检测质子化水中的质子信号。 对核糖核酸酶A和胰凝乳蛋白酶进行了初步研究。 用于rna酶 在10 mM浓度下， 可以很容易地在氚光谱中检测到。 线在13.736， 13.162和12.470 ppm的同位素位移分别为-0.177、-0.116和-0.099 ppm的。 因此，似乎有一个一般的相关性， 化学位移和同位素位移。 在糜蛋白酶中，样品仅 2 mM的质子共振，并且在受抑制的细胞中在约18.5ppm的质子共振。 酶在氚光谱中不可见。 这个实验将 重复检测，以优化检测灵敏度， 如果需要的话，具有更长的获取时间。 另一个实验是 计划进行T-15 N相关性，以检测T 和葡萄球菌核酸酶样品中的15 N。

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.