(c)Analogous imidazol in ones autoxidize spontaneously(26).(d)The proposed cyclization is isosteric with the known tendency for Asn-Gly sequences to cyclize to imides (27). Glycine is by far the best nucleophile in such cyclizations because of its minimal steric hindrance, and Gly67 is conserved in all known mutants of GFP that retain fluorescence. (e) Electrospray mass spectra indicate that anaerobically preformed GFP loses only 1 ± 4 Da upon exposure to air, consistent with the predicted loss of two hydrogens (22). This implies that the dehydration (−18 Da) must already have occurred anaerobically and must precede oxidation. (f) Reid & Flynn (23) have extensively characterized the kinetics of in vitro refolding of GFP from bacterial inclusion bodies with no chromophore, urea-denatured protein with a mature chromophore, and denatured protein with a chromophore reduced by dithionite. Renaturation was measuredbydevelopmentoffluorescenceandresistancetotrypsinattack