Vibrational Stark Effect in Human Aldose Reductase

描述(申请人提供)：人类醛糖还原酶(HALR2)在高血糖条件下将葡萄糖还原为山梨醇，被认为是糖尿病长期健康影响的原因，包括白内障和肾病。寻找不与结构相似的蛋白质-人醛还原酶(HALR1)结合的hALR2的强抑制剂的关键之一是测量抑制剂结合部位的静电场。在这样一个复杂的系统中，将使用hALR2与含有氰基官能团的抑制剂结合的振动斯塔克效应(VSE)光谱来测量这些场，氰基官能团的振动吸收频率位于光谱中没有蛋白质吸收的区域。首先，将合成一系列的抑制剂分子，其中氰基的位置围绕蛋白质活性部位的疏水口袋移动。其次，将获得蛋白质-抑制物复合体的晶体结构。最后，活性部位附近的氨基酸残基将发生突变，从而改变探针周围的局部静电场。然后对活性部位结合了抑制剂的蛋白质突变体进行VSE光谱分析。结果将与hALR2中当前静电场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.