Abstract
Coupling with ATP hydrolysis and cooperating with GroES, the double ring chaperonin GroEL assists the folding of other proteins. Here we report novel GroEL-GroES complexes formed in fluoroberyllate (BeF(x)) that can mimic the phosphate part of the enzyme-bound nucleotides. In ATP, BeF(x) stops the functional turnover of GroEL by preventing GroES release and produces a symmetric 1:2 GroEL-GroES complex in which both GroEL rings contain ADP.BeF(x) and an encapsulated substrate protein. In ADP, the substrate protein-loaded GroEL cannot bind GroES. In ADP plus BeF(x), however, it can bind GroES to form a stable 1:1 GroEL-GroES complex in which one of GroEL rings contains ADP.BeF(x) and an encapsulated substrate protein. This 1:1 GroEL-GroES complex is converted into the symmetric 1:2 GroEL-GroES complex when GroES is supplied in ATP plus BeF(x). Thus, BeF(x) stabilizes two GroEL-GroES complexes; one with a single folding chamber and the other with double folding chambers. These results shed light on the intermediate ADP.P(i) nucleotide states in the functional cycle of GroEL.
Highlights
Chaperonins are a class of molecular chaperones that promote protein folding in the cell and are found in bacteria, chloroplasts, mitochondria, Archaea, and eukaryotic cytosol [1, 2]
Current understanding of the major productive pathway of an ATP hydrolysis-coupled GroEL function starting from the substrate protein-loaded GroEL is as follows. (i) For cis-ternary complex formation, the binding of ATP to the substrate protein-loaded heptamer ring of GroEL permits the binding of GroES to this ring generating an enlarged, closed central cavity in
According to the above scheme, the step (ii), the trans-ring activation involves the hydrolysis of ATP in the cis-ATP complex to generate a transient intermediate cis-ADP1⁄7Pi complex, which is followed by rapid Pi release [15]
Summary
Proteins and Reagents—BeCl2 was purchased from Aldrich. NaF and AlCl3 were obtained from Wako (Osaka, Japan). Malate dehydrogenase (porcine), proteinase K, ATP, ADP, and NADH were obtained from Roche Applied Science. Calcium-depleted bovine ␣-lactalbumin (Type III), pyruvate kinase, lactate dehydrogenase, and hexokinase were from Sigma. Cy3-NHS (Fluorolink Cy3 monofunctional dye) was from Amersham Biosciences. GroEL, GroES, and bovine mitochondrial rhodanese were purified as described [20]. GroEL purified by the procedures including gel-filtration column chromatography in the presence of
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
