How Is cis-trans Isomerization Controlled in Dronpa Mutants? A Replica Exchange Molecular Dynamics Study Samuel L. C. Moors, Servaas Michielssens, Cristina Flors, † Peter Dedecker, Johan Hofkens, and Arnout Ceulemans* Department of Chemistry and INPAC Institute for Nanoscale Physics and Chemistry, K. U. LeuVen, LeuVen, Belgium Received February 1, 2008 Abstract: The reversibly photoactivatable green fluorescent protein analog Dronpa holds great promise as a marker for various new cellular imaging applications. Using a replica exchange method which combines both Hamiltonian and temperature exchanges, the ground-state dynamics of Dronpa and two mutants with increased switching kinetics, Val157Gly and Met159Thr, were compared. The dominant chromophore state was found to be the cis isomer in all three proteins. The simulation data suggest that both mutations strongly increase the chromophore flexibility and cis-trans isomerization rate. We identify three key amino acids, Val157, Met159, and Phe173, which are able to impede the bottom hula-twist transition path, depending on their position and rotameric state. We believe our insights will help to understand the switching process and provide useful information for the design of new variants with improved fluorescence properties. Introduction Dronpa, a monomeric mutant of a fluorescent protein from the coral Pectiniidae Echinophyllia sp. SC22, sharing 81% sequence identity with the fluorescent protein KikG from the coral FaVia faVus, emits 518 nm light with a high fluorescence quantum yield (Φ FL ) 0.85). 1,2 At neutral or basic pH, Dronpa can be photoswitched from a bright (fluorescent) state to a dark (nonfluorescent) state by intense 488 nm light. The photoswitched dark state has a half- lifetime of 14 h. 3 Illumination with 405 nm light efficiently switches the dark state back to the bright state. This on-off switching process is highly reversible (>100 cycles), 4 opening up a host of new applications including dynamic optical labeling and tracking of proteins, organelles, or cells and detection of protein interactions using high-resolution imaging. 5,6 Recently, through semirandom mutagenesis, Ando et al. discovered two Dronpa mutants, Dronpa Met159Thr (Dronpa- 2) and Dronpa Val157Ile/Met159Ala (Dronpa-3), which display increased photoinduced and spontaneous switching kinetics. 7 Compared to Dronpa, both mutants can be turned off more efficiently with 488 nm light and returned more quickly to their emissive states in the dark but have much lower fluorescence quantum yields (Φ FL ) 0.33 and 0.28 respectively). Ensemble and single-molecule fluorescence experiments suggested increased conformational freedom of the chromophore in Dronpa-2 and Dronpa-3 and formation of a dark intermediate state arising from cis-trans isomer- ization. 8 Independently, Stiel et al. 3 also discovered Dronpa- 2, together with another fast-switching mutant, Dronpa Val157Gly (rsFastLime), with a slightly decreased fluores- cence quantum yield (Φ FL ) 0.77). Both variants were designed by structural comparison of Dronpa with asFP595 from the sea anemone Anemonia sulcata. Like Dronpa, asFP595 is a reversibly photoactivatable protein. With only four differing residues, the immediate chromophore environ- ments of asFP595 and Dronpa are very similar. Unlike the Dronpa chromophore, which adopts a fluorescent cis con- formation, the equilibrated asFP595 chromophore is in a nonfluorescent trans conformation. The residues Val157 and Ser142 in Dronpa are replaced in asFP595 at the equivalent positions by serine and alanine, respectively. As a result, the favorable H-bond between the cis chromophore and * Corresponding author tel.: (32)16/32.73.63; fax: (32)16/ 32.79.92; e-mail: Arnout.Ceulemans@chem.kuleuven.be. † Present address: School of Chemistry, University of Edinburgh, UK. J. Chem. Theory Comput. 2008, 4, 1012–1020 1012 10.1021/ct8000359 CCC: $40.75  2008 American Chemical Society Published on Web 05/20/2008