On excited state reaction path in reversibly switchable fluorescent proteins

TL;DR

This study uses advanced QM/MM simulations to elucidate the molecular mechanisms behind spectral tuning and photo-switching in five closely related reversibly switchable fluorescent proteins, linking structural changes to photophysical properties.

Contribution

It provides a detailed mechanistic understanding of spectral variations and switching behavior in RSFPs through combined molecular dynamics and quantum chemistry calculations.

Findings

Models reproduce absorption and emission trends with limited blue-shifts.

Charge-transfer patterns relate to hydrogen-bond network rearrangements.

Conical intersection topography correlates with photophysical properties.

Abstract

A set formed by five reversibly-switchable fluorescent proteins (RSFPs) display spread over 40~nm in absorption maxima and only 18~nm in emission. The five proteins -- Dronpa, rsFastLime, rsKame, Padron(anionic form) and bsDronpa -- carry exactly the same chromophore and differ just in a few mutations. Thus they form an ideal set for mechanistic investigation. Starting with the results of molecular dynamics simulations we use QM/MM calculations to investigate the effects controlling the spectral tuning . In this contribution we show that the models, which are based on CASPT2//CASSCF level of QM theory, reproduce the observed absorption trend with only a limited blue-shift of 4.5 kcal/mol and emission trend with even smaller blue-shift of 1.5 kcal/mol. Using CASSCF QM/MM calculations we analyze the chromophore's charge-transfer patterns during the absorption and emission, which, in turn trigger a cascade of hydrogen-bond-network rearrangements indicating a preparation to an isomerization event. We also show how the contribution of individual aminoacids to the chromophore conformational changes correlates with spectral tuning of the absorption and emission. Furthermore, we identify how the conical intersection topography correlates with protein's photophysical properties. In conclusion, we establish a detailed mechanistic explanation of variations in photo-switching speed as well as higher sensitivity of RSFPs to mutation observed for light absorption relative to light emission.