CHAPERONIN-MEDIATED FOLDING OF GREEN FLUORESCENT PROTEIN

Citation
Y. Makino et al., CHAPERONIN-MEDIATED FOLDING OF GREEN FLUORESCENT PROTEIN, The Journal of biological chemistry, 272(19), 1997, pp. 12468-12474
Citations number
36
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0021-9258
Volume
272
Issue
19
Year of publication
1997
Pages
12468 - 12474
Database
ISI
SICI code
0021-9258(1997)272:19<12468:CFOGFP>2.0.ZU;2-V
Abstract
Chaperonin-mediated folding of green fluorescent protein (GFP) was exa mined by real time monitoring of recovery of fluorescence and by gel f iltration high-performance liquid chromatography, Acid-denatured GFP c an fold spontaneously upon dilution into the neutral buffer, When Esch erichia coli GroEL/ES was present, folding of GFP was arrested. Foldin g was resumed by subsequent addition of 100 mu m or 1 mM ATP, and nati ve GFP was regenerated to 100% yield. When folding was resumed by 10 m u 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 o f the GroEL ring underneath GroES (cis-folding), Because the overall r ates of GroEL/ES, ATP-mediated GFP folding were all similar to that of spontaneous folding, it was concluded that cis-folding proceeded as f ast as spontaneous folding, The GroEL/ES-bound native GFP was observed only when both GroES and ATP (but not ADP) were present in the foldin g mixture, Holo-chaperonin from Thermus thermophilus, which was purifi ed as a cpn60/10 complex, exhibited the similar cis-folding, Consisten tly, ATP-dependent exchange of cpn10 in the holo-chaperonin with free cpn10 was observed.