Circularly permuted variants of the green fluorescent protein

Folding of the green fluorescent protein (GFP) from Aequorea victoria is ch aracterized by autocatalytic formation of its p-hydroxybenzylideneimidazoli done chromophore, which is located in the center of an 11-stranded beta-bar rel. We have analyzed the in vivo folding of 20 circularly permuted variant s of GFP and find a relatively low tolerance towards disruption of the poly peptide chain by introduction of nea termini. All permuted variants with te rmini in strands of the beta-barrel and about half of the variants with ter mini in loops lost the ability to form the chromophore. The thermal stabili ty of the permuted GFPs with intact chromophore is very similar to that of the wild-type, indicating that chromophore-side chain interactions strongly contribute to the extraordinary stability of GFP. (C) 1999 Federation of E uropean Biochemical Societies.