Vibrational Stark Effect in Human Aldose Reductase

描述（由申请人提供）：人醛糖还原酶（hALR 2）在高血糖条件下将葡萄糖还原为山梨糖醇，并且被认为是糖尿病（包括白内障和肾病）的长期健康影响的原因。找到不与结构相似的蛋白质人醛还原酶（hALR 1）结合的hALR 2强抑制剂的一个关键是测量抑制剂结合位点的静电场。在这样一个复杂的系统中测量这些场将使用与抑制剂结合的hALR 2的振动斯塔克效应（VSE）光谱来完成，所述抑制剂含有氰基官能团，其在没有蛋白质吸收的光谱区域中具有振动吸收频率。首先，将合成一系列抑制剂分子，其中氰基的位置围绕蛋白质活性位点的疏水口袋移动。其次，将获得蛋白质-抑制剂复合物的晶体结构。最后，活性位点附近的氨基酸残基将被突变以改变探针周围的局部静电场。然后将对具有与活性位点结合的抑制剂的蛋白质突变体进行VSE光谱分析。将结果与hALR 2中静电场的当前计算模型的预测进行比较。

DESCRIPTION (provided by applicant): Human aldose reductase (hALR2) reduces glucose to sorbitol under hyperglycemic conditions, and is thought to be a cause of long-term health effects of diabetes including cataracts and nephropathy. One key to finding strong inhibitors of hALR2 that do not also bind to a structurally similar protein, human aldehyde reductase (hALR1), is to measure the electrostatic field of the inhibitor binding site. Measuring these fields in such a complicated system will be done using vibrational Stark effect (VSE) spectroscopy of hALR2 bound with inhibitors which contain a cyano functional group, which has a vibrational absorption frequency in a region of the spectrum that is free of protein absorption. First, a series of inhibitor molecules will be synthesized in which the location of the cyano group is moved around the hydrophobic pocket of the protein active site. Second, a crystal structure of the protein-inhibitor complex will be obtained. Finally, amino acid residues near the active site will be mutated to alter the local electrostatic field surrounding the probe. VSE spectroscopy will then be conducted on protein mutants with inhibitor bound to the active site. Results will be compared to predictions from current computational models of electrostatic fields in hALR2.