CRYSTAL-STRUCTURE AND PHOTODYNAMIC BEHAVIOR OF THE BLUE EMISSION VARIANT Y66H Y145F OF GREEN FLUORESCENT PROTEIN/

The crystal structure of a blue emission variant (Y66H/Y145F) of the A equorea victoria green fluorescent protein has been determined by mole cular replacement and the model refined. The crystallographic R-factor is 18.1% for all data from 20 to 2.1 Angstrom, and the model geometry is excellent. The chromophore is non-native and is autocatalytically generated from the internal tripeptide Ser65-His66-Gly67. The final el ectron density maps indicate that the formation of the chromophore is complete, including 1,2 dehydration of His66 as indicated by the plana rity of the chromophore. The chromophore is in the cis conformation, w ith no evidence for any substantial fraction of the trans configuratio n or uncyclized apoprotein, and is well-shielded from bulk solvent by the folded protein. These characteristics indicate that the machinery for production of the chromophore from a buried tripeptide unit is not only intact but also highly efficient in spite of a major change in c hromophore chemical structure. Nevertheless, there are significant rea rrangements in the hydrogen bond configuration around the chromophore as compared to wild-type, indicating flexibility of the active site. p H titration of the intact protein and the chromopeptide (pK(a1) = 4.9 +/- 0.1, pK(a2) = 12.0 +/- 0.1) suggests that the predominant form of the chromophore in the intact protein is electrically neutral. In cont rast to the wild-type protein [Chattoraj, M., King, B. A., Bubliti, G. U., & Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A., 8362-8367], femtosecond fluorescence up-conversion spectroscopy of the intact prot ein and a partially deuterated form strongly suggests that excited-sta te proton transfer is not coupled to fluorescence emission.