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 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.
Makino
Y
Y
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226, Japan.
Amada
K
K
Taguchi
H
H
Yoshida
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D018835:Chaperonin 10 / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism; D018833:Chaperonins / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D013824:Thermus; D016963:Thermus thermophilus
D018835:Chaperonin 10; D018834:Chaperonin 60; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D018833:Chaperonins