Reversibly switchable fluorescent proteins: “the fair switch project”

Abstract

AbstractFluorescent proteins (FPs) have transformed cell biology through their use in fluorescence microscopy, enabling precise labeling of proteins via genetic fusion. A key advancement is altering primary sequences to customize their photophysical properties for specific imaging needs. A particularly notable family of engineered mutants is constituted by Reversible Switching Fluorescent Proteins (RSFPs), i.e. variant whose optical properties can be toggled between a bright and a dark state, thereby adding a further dimension to microscopy imaging. RSFPs have strongly contributed to the super-resolution (nanoscopy) revolution of optical imaging that has occurred in the last 20 years and afforded new knowledge of cell biochemistry at the nanoscale. Beyond high-resolution applications, the flexibility of RSFPs has been exploited to apply these proteins to other non-conventional imaging schemes such as photochromic fluorescence resonance energy transfer (FRET). In this work, we explore the origins and development of photochromic behaviors in FPs and examine the intricate relationships between structure and photoswitching ability. We also discuss a simple mathematical model that accounts for the observed photoswitching kinetics. Although we review most RSFPs developed over the past two decades, our main goal is to provide a clear understanding of key switching phenotypes and their molecular bases. Indeed, comprehension of photoswitching phenotypes is crucial for selecting the right protein for specific applications, or to further engineer the existing ones. To complete this picture, we highlight in some detail the exciting applications of RSFPs, particularly in the field of super-resolution microscopy.