EVOLUTION AND CHARACTERIZATION OF A NEW REVERSIBLY PHOTOSWITCHABLE CHROMOPROTEIN
We report the molecular engineering and spectral characterization of a new reversibly photoswitchable chromoprotein, B11 a variant of thermostable green protein (TGP) that has been evolved for favorable solubility, thermostability, and enhanced crystallization properties. Its 2.5 \xc5 joint X-ray and neutron structure shows the location of critical hydrogen atoms, and reveals the position, orientation, and protonation states of solvent molecules in the chromophore and surrounding amino acids, which have not been ascertained to date from current X-ray structures. We report 1.65 \xc5 ground state and light-induced state X-ray crystal structures of B11, which differs from TGP by four amino acids and has a weak fluorescence in its ground state, and these structures are compared to the wild-type TGP structure. These structures will enhance future engineering of new and novel fluorescent proteins.
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