Crystal structure and refolding properties of the mutant F99S/M153T/V163A of the Green Fluorescent Protein

The mutant F99S/M153T/V163A of the Green Fluorescent Protein (c3-GFP) has s pectral characteristics similar to the wild-type GFP, but it is 42-fold mor e fluorescent in vivo, Here, we report the crystal structure and the refold ing properties of c3-GFP and compare them with those of the less fluorescen t wt-GFP and S65T mutant, The topology and the overall structure of c3-GFP is similar to the wild-type GFP. The three mutated residues, Ser99, Thr153, and Ala163, lie on the surface of the protein in three diffel ent beta -st rands, The side chains of Ser99 and Thr153 are exposed to the solvent, wher eas that of Ala163 points toward the interior of the protein. No significan t deviation from the structure of the wild-type molecule is found around th ese positions, and there is not clear evidence of any distortion in the pos ition of the chromophore or of the surrounding residues induced by the muta ted amino acids. In vitro refolding experiments on urea-denatured c3-GFP re veal a renaturation behavior similar to that of the S65T molecule, with kin etic constants of the same order of magnitude. We conclude that the higher fluorescence activity of c3-GFP can be attributed neither to particular str uctural features nor to a faster folding process, as previously proposed. P roteins 2000;41:429-437, (C) 2000 Wiley-Liss, Inc.