Abstract
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 microM or 1 mM ATP, and native GFP was regenerated to 100% yield. When folding was resumed by 10 microM 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.
Highlights
Members of the chaperonin family play an essential role in facilitating folding in the cytosol of both prokaryotes and eukaryotes [1,2,3,4,5,6]
Green fluorescent protein (GFP) has the advantage for the study of protein folding, that is, one can readily monitor the folding in real time using fluorescence as a marker of recovery of native structure
When molar excess GroEL/ES over GFP was present in the dilution buffer, unfolded GFP was captured by GroEL/ES almost completely upon dilution (Fig. 1A, filled arrowhead) and stayed bound to GroEL/ES without producing native GFP
Summary
Members of the chaperonin family play an essential role in facilitating folding in the cytosol of both prokaryotes and eukaryotes [1,2,3,4,5,6]. Binding of substrate polypeptide occurs exclusively to the GroEL ring not occupied by GroES, as observed by electron micrograph (trans-complex) [12, 13]. It rebinds to the trans GroEL ring to regenerate transcomplex, or it completes folding by itself in the medium if conditions are suitable for spontaneous folding. When GroES is released, it rebinds to either one of two GroEL rings If it binds to the GroEL ring not containing polypeptide, trans-complex is regenerated. For cis-folding to occur, cpn should be released from one cpn ring and rebind to the cpn ring of the opposite side where substrate polypeptide is already bound. Can Thermus holo-chaperonin mediate cis-folding, and does the release-rebinding of cpn really happen? Can Thermus holo-chaperonin mediate cis-folding, and does the release-rebinding of cpn really happen? Here, taking advantage of GFP as a substrate protein, we have tried to answer some of these questions
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