  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.
