Green fluorescent protein variants fold differentially in prokaryotic and eukaryotic cells

Better-folding Green Fluorescent Protein (GFP) mutants selected from bacter ial screenings are commonly used in widely different cellular environments. However, it is unclear if the folding efficiency of GFPs is invariant in d ifferent cell types. In this work, we have analysed the folding properties of GFP variants in bacteria versus mammalian cells. Remarkably, S65T was fo und to fold at comparable levels with the wild type GFP in bacteria, but at 10-fold lower levels in mammalian cells. On the other hand, Bex1 folded 3- 4 times better than the wtGFP or S65T in E. coli, and 10-20-fold or more th an YS-fold better, respectively, in mammalian cells. The Veri mutant demons trated similar properties to Bex1. No evidence of differential GFP unfoldin g in vivo or of preferential degradation of unfolded GFP molecules was foun d. Moreover, no relationship between GFP folding efficiency and expression levels, or protein stability was detected. Trivial Aconfounding factors, li ke GFP unfolding caused by different pH or fluorescence quenching due to mo lecular crowding, were also excluded. In summary, our results demonstrate t hat specific GFP variants follow different folding trajectories in mammalia n versus bacterial cells. The specificity of this differential folding supp orts a role of chaperones in guiding the folding of GFP in vivo. (C) 2001 W iley-Liss, Inc.