Three-Dimensional Structure of the S148F Mutant of Blue-to-Red Fluorescent Timer mRubyFT

Abstract

Genetically encoded fluorescent timers change their fluorescence color over time and are used for tracking the age of proteins, cell organelles, and cells, visualizing the protein trafficking, monitoring the promoter activity, and in vivo labeling of two neuronal populations activated in two different episodes. The recently reported genetically encoded blue-to-red mRubyFT fluorescent timer showed improved brightness and enhanced photostability in mammalian cells, was applied to label cytoskeleton proteins, and retained the timer properties in mammalian cells. Although the structure of the red form of this timer was determined at high resolution, the mechanism of the blue-to-red transition is not fully understood. The single-point S148F mutant of the mRubyFT fluorescent timer was constructed. This mutation site is supposed to be critical for the stabilization of the blue form of the chromophore. The structure of the mRubyFT^S148F blue form was determined at 1.6 Å resolution and compared with the structure of the red form of mRubyFT.