Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

详细信息    查看全文

• 作者：Sam Duw茅 ; Elke De Zitter ; Vincent Gielen ; Benjamien Moeyaert ; Wim Vandenberg ; Tim Grotjohann ; Koen Clays ; Stefan Jakobs ; Luc Van Meervelt ; Peter Dedecker
• 刊名：ACS Nano
• 出版年：2015
• 出版时间：October 27, 2015
• 年：2015
• 卷：9
• 期：10
• 页码：9528-9541
• 全文大小：893K
• ISSN：1936-086X

文摘

鈥淪mart fluorophores鈥? such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 掳C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of 鈭?0 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the 鈥渙n鈥? and 鈥渙ff鈥?conformation we were able to confirm the presence of a cis鈥搕rans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded 鈥渟mart fluorophores鈥?can be readily optimized for biological performance and provides a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins.