Ravikumar Yuvaraj, Nadarajan Saravanan Prabhu, Lee Chong-Soon, Rhee Jin-Kyu, Yun Hyung-Don
School of Biotechnology, Department of Biochemistry, Yeungnam University, Gyeongsan 712-749, Republic of Korea.
Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea.
J Microbiol Biotechnol. 2015 Apr;25(4):503-10. doi: 10.4014/jmb.1409.09035.
The iLOV protein belongs to a family of blue-light photoreceptor proteins containing a lightoxygen- voltage sensing domain with a noncovalently bound flavin mononucleotide (FMN) as its chromophore. Owing to advantages such as its small size, oxygen-independent nature, and pH stability, iLOV is an ideal candidate over other reporter fluorescent proteins such as GFP and DsRed. Here, for the first time, we describe the feasibility of applying LOV domain-based fluorescent iLOV as a metal sensor by measuring the fluorescence quenching of a protein with respect to the concentration of metal ions. In the present study, we demonstrated the inherent copper sensing property of the iLOV protein and identified the possible amino acids responsible for metal binding. The fluorescence quenching upon exposure to Cu(2+) was highly sensitive and exhibited reversibility upon the addition of the metal chelator EDTA. The copper binding constant was found to be 4.72 ± 0.84 micrometer. In addition, Cu(2+)-bound iLOV showed high fluorescence quenching at near physiological pH. Further computational analysis yielded a better insight into understanding the possible amino acids responsible for Cu(2+) binding with the iLOV protein.
iLOV蛋白属于蓝光感光蛋白家族，其包含一个光氧电压传感结构域，该结构域以非共价结合的黄素单核苷酸（FMN）作为发色团。由于iLOV具有体积小、不依赖氧气以及pH稳定性等优点，相较于其他报告荧光蛋白如绿色荧光蛋白（GFP）和红色荧光蛋白（DsRed），它是一个理想的选择。在此，我们首次通过测量蛋白质荧光相对于金属离子浓度的淬灭情况，描述了将基于LOV结构域的荧光蛋白iLOV用作金属传感器的可行性。在本研究中，我们展示了iLOV蛋白固有的铜传感特性，并确定了可能负责金属结合的氨基酸。暴露于Cu(2+)时的荧光淬灭非常敏感，并且在添加金属螯合剂乙二胺四乙酸（EDTA）后表现出可逆性。发现铜结合常数为4.72±0.84微摩尔。此外，结合Cu(2+)的iLOV在接近生理pH值时表现出高荧光淬灭。进一步的计算分析有助于更深入地了解可能负责iLOV蛋白与Cu(2+)结合的氨基酸。
