Chaperonin-mediated folding of green fluorescent protein

Chaperonin-mediated folding of green fluorescent protein (GFP) was examined by real time monitoring of recovery of fluorescence and by gel filtration high-performance liquid chromatography, Acid-denatured GFP can fold spontaneously upon dilution into the neutral buffer, When Escherichia coli GroEL/ES was present, folding of GFP was arrested. Folding was resumed by subsequent addition of 100 mu m or 1 mM ATP, and native GFP was regenerated to 100% yield. When folding was resumed by 10 mu M ATP (1.4 mol/mol GroEL subunit), about 60% of GFP recovered native structure, and one-half of them (30%) was found to be still bound to GroEL/ES, indicating the occurrence of folding in the central cavity of the GroEL ring underneath GroES (cis-folding), Because the overall rates of GroEL/ES, ATP-mediated GFP folding were all similar to that of spontaneous folding, it was concluded that cis-folding proceeded as fast as spontaneous folding, The GroEL/ES-bound native GFP was observed only when both GroES and ATP (but not ADP) were present in the folding mixture, Holo-chaperonin from Thermus thermophilus, which was purified as a cpn60/10 complex, exhibited the similar cis-folding, Consistently, ATP-dependent exchange of cpn10 in the holo-chaperonin with free cpn10 was observed.