Department of Biomedical Sciences, Graduate School of Medicine , Korea University , Seoul 02841 , Republic of Korea.
Department of Applied Chemistry , Kookmin University , Seoul 02707 , Republic of Korea.
J Agric Food Chem. 2019 Oct 30;67(43):12037-12043. doi: 10.1021/acs.jafc.9b05473. Epub 2019 Oct 17.
Despite remarkable contribution of green fluorescent protein and its variants for better understanding of various biological functions, its application for anaerobic microorganisms has been limited because molecular oxygen is essential for chromophore formation. To overcome the limitation, we engineered a plant-derived light, oxygen, or voltage (LOV) domain containing flavin mononucleotide for enhanced spectral properties. The resulting LOV variants exhibited improved fluorescence intensity (20 and 70% higher for SH3 and 70% for BR1, respectively) compared to iLOV, an LOV variant isolated in a previous study, and the quantum yields of the LOV variants (0.40 for SH3 and 0.45 for BR1) were also improved relative to that of iLOV ( = 0.37). In addition to fluorescence intensity, the identified mutations of SH3 enabled an improved thermostability of the protein. The engineered LOV variants with enhanced spectral properties could provide a valuable tool for fluorescent molecular probes under anaerobic conditions.
尽管绿色荧光蛋白及其变体在更好地理解各种生物功能方面做出了显著贡献，但由于生色团的形成需要分子氧，因此其在厌氧微生物中的应用受到限制。为了克服这一限制，我们构建了一种含有黄素单核苷酸的植物衍生的光、氧或电压 (LOV) 结构域，以增强光谱特性。与之前研究中分离的 LOV 变体 iLOV 相比，所得 LOV 变体的荧光强度分别提高了 20%和 70%（对于 SH3 和 BR1 分别提高了 70%），并且 LOV 变体的量子产率（对于 SH3 为 0.40，对于 BR1 为 0.45）也高于 iLOV（ = 0.37）。除了荧光强度之外，SH3 的鉴定突变还使蛋白质的热稳定性得到改善。具有增强光谱特性的工程 LOV 变体可以为厌氧条件下的荧光分子探针提供有价值的工具。
