Electrostatic fields near the active site of human aldose reductase: 1. New inhibitors and vibrational stark effect measurements
Electrostatic fields near the active site of human aldose reductase: 1. New inhibitors and vibrational stark effect measurements
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DOI：
10.1021/bi701708u
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发表时间：
2008-02-12
期刊：
影响因子：
2.9
通讯作者：
Boxer, Steven G.
中科院分区：
文献类型：
--
作者：
Webb, Lauren J.;Boxer, Steven G.
采用振动斯塔克效应光谱法测定了人醛糖还原酶（hALR2）活性位点疏水区的静电场。设计并合成了一种新的hALR2抑制剂，该抑制剂含有一个腈探针，其Stark调谐速率为0.77 cm(-1)（MV/cm）。根据静电计算、腈附近氢键可能引起的并发症以及与结构非常相似的人醛还原酶活性位点的比较，选择了腈官能团附近氨基酸残基的突变位点。当与这些突变的蛋白质结合时，腈探针的吸收能量的变化被用来量化蛋白质静电场的扰动。观察到的静电场变化可达-10 MV/cm。将测量的静电场与基于连续体静电计算的预测结果进行了比较，揭示了对计算策略的实质性修改是必要的。考虑了氨基酸侧链氢键对腈探针的影响，讨论了振动斯塔克效应光谱在配体结合和生物功能研究中的应用。
Vibrational Stark effect spectroscopy was used to measure electrostatic fields in the hydrophobic region of the active site of human aldose reductase (hALR2). A new hALR2 inhibitor was designed and synthesized that contains a nitrile probe with a Stark tuning rate of 0.77 cm(-1)(MV/cm). Mutations to amino acid residues in the vicinity of the nitrile functional group were selected based on electrostatics calculations, possible complications from hydrogen bonds near the nitrile, and comparison with the active site of human aldehyde reductase, whose structure is very similar. Changes in the absorption energy of the nitrile probe when bound to those mutated proteins were then used to quantify perturbations to the protein's electrostatic field. Electrostatic field changes as large as -10 MV/cm were observed. Measured electrostatic fields were compared to predictions based on continuum electrostatics calculations, revealing that substantial modifications to the calculation strategy are necessary. The effects of hydrogen bonding of amino acid side chains to the nitrile probe are considered, and applications of vibrational Stark effect spectroscopy to investigations of ligand binding and biological function are discussed.