Subach Oksana M, Gundorov Illia S, Yoshimura Masami, Subach Fedor V, Zhang Jinghang, Grüenwald David, Souslova Ekaterina A, Chudakov Dmitriy M, Verkhusha Vladislav V
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Chem Biol. 2008 Oct 20;15(10):1116-24. doi: 10.1016/j.chembiol.2008.08.006.
We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Förster resonance energy transfer applications.
我们采用了一种红色发色团形成途径，其中阴离子红色发色团由中性蓝色中间体形成，以此提出一种合理的设计策略，用于开发具有基于酪氨酸发色团的蓝色荧光蛋白。该策略应用于不同遗传背景的红色荧光蛋白，如TagRFP、mCherry、HcRed1、M355NA和mKeima，它们都被转化为蓝色探针。通过随机诱变对TagRFP的蓝色变体进行进一步改进，得到了一种增强型单体蛋白mTagBFP，其特点是与发色团中含有组氨酸的蓝色荧光蛋白相比，亮度显著更高、发色团成熟更快且pH稳定性更高。详细的生化和光化学分析表明，mTagBFP是用于多色和寿命成像的真正单体蛋白标签，也是Förster共振能量转移应用中绿色荧光蛋白的出色供体。
