Dean Kevin M, Lubbeck Jennifer L, Davis Lloyd M, Regmi Chola K, Chapagain Prem P, Gerstman Bernard S, Jimenez Ralph, Palmer Amy E
BioFrontiers Institute, University of Colorado, Boulder, CO, USA.
Integr Biol (Camb). 2015 Feb;7(2):263-73. doi: 10.1039/c4ib00251b.
Fluorescent proteins offer exceptional labeling specificity in living cells and organisms. Unfortunately, their photophysical properties remain far from ideal for long-term imaging of low-abundance cellular constituents, in large part because of their poor photostability. Despite widespread engineering efforts, improving the photostability of fluorescent proteins remains challenging due to lack of appropriate high-throughput selection methods. Here, we use molecular dynamics guided mutagenesis in conjunction with a recently developed microfluidic-based platform, which sorts cells based on their fluorescence photostability, to identify red fluorescent proteins with decreased photobleaching from a HeLa cell-based library. The identified mutant, named Kriek, has 2.5- and 4-fold higher photostability than its progenitor, mCherry, under widefield and confocal illumination, respectively. Furthermore, the results provide insight into mechanisms for enhancing photostability and their connections with other photophysical processes, thereby providing direction for ongoing development of fluorescent proteins with improved single-molecule and low-copy imaging capabilities.
荧光蛋白在活细胞和生物体中具有出色的标记特异性。不幸的是，就低丰度细胞成分的长期成像而言，它们的光物理性质仍远不理想，这在很大程度上是由于其光稳定性较差。尽管进行了广泛的工程改造，但由于缺乏合适的高通量筛选方法，提高荧光蛋白的光稳定性仍然具有挑战性。在这里，我们将分子动力学引导的诱变与最近开发的基于微流控的平台相结合，该平台根据细胞的荧光光稳定性对细胞进行分类，以从基于HeLa细胞的文库中鉴定出光漂白减少的红色荧光蛋白。鉴定出的突变体名为Kriek，在宽场和共聚焦照明下，其光稳定性分别比其亲本mCherry高2.5倍和4倍。此外，这些结果为增强光稳定性的机制及其与其他光物理过程的联系提供了见解，从而为正在进行的具有改进的单分子和低拷贝成像能力的荧光蛋白的开发提供了方向。
