5DTL

Crystal structure of mEos2-A69T fluorescent protein

5DTL の概要

• エントリーDOI: 10.2210/pdb5dtl/pdb
• 分子名称: Green to red photoconvertible GFP-like protein EosFP (2 entities in total)
• 機能のキーワード: fluorescent protein, meos2, blinking
• 由来する生物種: Lobophyllia hemprichii (Lobed brain coral)
• タンパク質・核酸の鎖数: 12
• 化学式量合計: 310721.21

構造登録者

Berardozzi, R.,Adam, V.,Martins, A.,Bourgeois, D. (登録日: 2015-09-18, 公開日: 2015-12-30, 最終更新日: 2024-10-23)

主引用文献

Berardozzi, R.,Adam, V.,Martins, A.,Bourgeois, D.
Arginine 66 Controls Dark-State Formation in Green-to-Red Photoconvertible Fluorescent Proteins.
J.Am.Chem.Soc., 138:558-565, 2016

PubMed Abstract: Photoactivated localization microscopy (PALM) is a powerful technique to investigate cellular nanostructures quantitatively and dynamically. However, the use of PALM for molecular counting or single-particle tracking remains limited by the propensity of photoconvertible fluorescent protein markers (PCFPs) to repeatedly enter dark states. By designing the single mutants mEos2-A69T and Dendra2-T69A, we completely swapped the blinking behaviors of mEos2 and Dendra2, two popular PCFPs. We combined X-ray crystallography and single-molecule microscopy to show that blinking in mEos2 and Dendra2 is largely controlled by the orientation of arginine 66, a highly conserved residue in Anthozoan PCFPs. The Arg66 side-chain conformation affects the bleaching and the on-to-off transition quantum yields, as well as the fraction of molecules entering long-lived dark states, resulting in widely different apparent blinking behaviors that largely modulate the efficiency of current blinking correction procedures. The present work provides mechanistic insight into the complex photophysics of Anthozoan PCFPs and will facilitate future engineering of bright and low-blinking variants suitable for PALM.

PubMed: 26675944
DOI: 10.1021/jacs.5b09923

実験手法

X-RAY DIFFRACTION (2.7 Å)