Exploring excited state structural dynamics of fluorescent proteins by using X-ray free electron laser (XFEL) towards advancement of cutting‐edge microscopy

Fluorescent proteins (FPs) have become a widely opted choice for protein labelling in living specimen. Some FPs show unique photoswitching properties (psFPs) with which advanced microscopic techniques such as super-resolution microscopy have been developed. Optimizing the photoswitching properties is crucial for further advancement of the microscopic technologies, and knowledge of molecular bases behind the photoswitching reactions is essential for rational design of improved psFPs. However, since the photoswitchings are excited processes proceeding in femto- to picosecond order, their structural dynamics are inaccessible by traditional synchrotron X-ray crystallography. In this project, we will address this unexplored information by employing the next generation X-ray source, X-ray free electron laser (XFEL). XFEL provides extremely short (10 fs), strong, and coherent X-ray pulses, which enables serial femtosecond X-ray crystallography (SFX). We will also employ ultrafast spectroscopy and QM/ MM simulation to acquire complementary information and integrate results with SFX, aiming full understanding of the molecular basis of the photoswitching processes. With these information, we will develop optimized psFPs for cutting-edge microscopy and apply them to progress biological imaging technologies.

Keywords:fluorescent protein, X-ray free electron laser, serial femtosecond X-ray crystallography, ultrafast spectroscopy, fluorescence microscopy, QM/MM modelling

Disciplines:Analytical spectrometry, Structural analysis, Cell signalling