A time-dependent DFT/molecular dynamics study of the proton-wire responsible for the red fluorescence in the LSSmKate2 protein

Fluorescent proteins (FP) have become a major topic in the recent biochemical research due to their applications as in vivo markers in biological systems. In particular, Red fluorescent proteins (RFP) present some advantages since they require less harmful radiations to be excited and show less light-scattering. In this paper, we are focusing on the LSSmKate2 protein, a RFP that, together with LSSmKate1 and mKeima, is well known for the outstanding difference between absorption and emission wavelengths, which is usually referred as Large Stokes Shift (LSS). It is commonly accepted that an excited state proton transfer accounts for the fluorescence observed in the three proteins. In this work, a molecular dynamics simulation of the LSSmKate2 protein has been carried out, and from different snapshots, a series of excited states have been calculated and analyzed. Our molecular dynamics simulation has proved the availability of the two-link proton-wire suggested by Piatkevich et al. and has furnished a new one-link relay, more prone to take place. The statistical treatment of the excited states can reproduce the electronic absorption spectrum in a reasonable way, and the analysis of the involved orbitals confirms that one absorption wavelength maximum corresponds to an acidification of the chromophore, regardless of the hydrogenbonded acceptor residue. All this work constitutes an important step in what should be a thorough and complete study of the photochemistry of the LSSproteins.