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首页> 外文期刊>RSC Advances >Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine
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Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine

机译：使用光谱报告子4-氰基-1-苯基丙氨酸探索血红素蛋白的局部溶剂化环境

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The vibrational reporter unnatural amino acid (UAA) 4-cyano- L -phenylalanine (pCNF) was genetically incorporated individually at three sites (5, 36, and 78) in the heme protein Caldanaerobacter subterraneus H-NOX to probe local hydration environments. The UAA pCNF was incorporated site-specifically using an engineered, orthogonal tRNA synthetase in E. coli . The ability of all of the pCNF-containing H-NOX proteins to form the ferrous CO, NO, or O _(2) ligated and unligated states was confirmed with UV-Vis spectroscopy. The solvation state at each site of the three sites of pCNF incorporation was assessed using temperature-dependent infrared spectroscopy. Specifically, the frequency–temperature line slope (FTLS) method was utilized to show that the nitrile group at site 36 was fully solvated and the nitrile group at site 78 was de-solvated (buried) in the heme pocket. The nitrile group at site 5 was found to be partially solvated suggesting that the nitrile group was involved in moderate strength hydrogen bonds. These results were confirmed by the determination of the X-ray crystal structure of the H-NOX protein construct containing pCNF at site 5.
机译：振动报告基因非天然氨基酸（UAA）4-氰基-L-苯丙氨酸（pCNF）分别在血红素蛋白质Caldanaerobacter subterraneus H-NOX的三个位点（5、36和78）上遗传整合，以探测局部水合环境。使用工程化的正交tRNA合成酶将UAA pCNF特异性结合在大肠杆菌中。通过UV-Vis光谱确认了所有含pCNF的H-NOX蛋白形成CO，NO或O _（2）亚铁键合和未键合状态的能力。使用温度依赖性红外光谱法评估pCNF引入的三个位点中每个位点的溶剂化状态。具体而言，使用频率-温度线斜率（FTLS）方法显示血红素袋中36位的腈基已完全溶解，而78位的腈基已被去溶剂化（埋藏）。发现位点5上的腈基部分溶剂化，表明腈基参与中等强度的氢键。通过测定第5位含有pCNF的H-NOX蛋白构建体的X射线晶体结构，证实了这些结果。

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