Leveraging the E222q Replacement to Generate Novel Reversibly Switchable Fluorescent Aequorea Victoria Proteins for Super-resolution Imaging

Reversibly photoswitchable fluorescent proteins (RSFPs) admirably combine the genetic encoding of fluorescence with the ability to repeatedly toggle between a bright and a dark state, adding a new temporal dimension to the fluorescence signal. Accordingly, in the last years RSFPs have paved the way to novel applications in cell imaging that rely on their reversible photoswitching, including many super-resolution techniques such as F-PALM, RESOLFT, and SOFI that provide nanoscale pictures of the living matter. Yet many RSFPs have been engineered by a rational approach only to a limited extent, in the absence of clear structure-property relationships that in most cases make the emergence of the photoswitching anecdotic. We have discovered that E222Q replacement is a single photoswitching mutation since it restores the intrinsic cis-trans photoisomerization properties of the chromophore in otherwise non-switchable Aequorea proteins of different color and mutation pattern (Q-RSFPs). Our findings link indissolubly photoswitching and Q222 presence, by a simple yet elegant scenario: largely twisted chromophore structures around the double bond (including hula-twist configurations) are uniquely stabilized by Q222 via H-bonds. Likely, these H-bonds subtly modulate the electronic properties of the chromophore, enabling the conical intersection that connects the excited cis to ground trans chromophore. Remarkably, analysis of photoswitching by fast spectroscopy revealed multiphase kinetics related to the peculiar protonation pattern of the protein. By applying E222Q to simple derivatives of the enhanced green fluorescent protein we generated a palette of green and yellow emitting Q-RSFPs that are tailored to several techniques addressing intracellular settings at nanoscale, such as photochromic FRET/anisotropy, SOFI, and F-PALM. This research has been partially funded by the project PRIN 2022RRFJC4 Novel protein-based Genetically-Encoded Fluorescent Indicators (GEFI) for Functional Super-Resolution Imaging of Biomolecular Activities in Living Cells [GEFInder]"