Double mutant MBP refolds at same rate in free solution as inside the GroEL/GroES chaperonin chamber when aggregation in free solution is prevented

• Published in: FEBS letters Vol. 585; no. 12; pp. 1969 - 1972
• Main Authors: Tyagi, Navneet K., Fenton, Wayne A., Deniz, Ashok A., Horwich, Arthur L.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 23.06.2011
• Subjects: Aggregation; Chaperonin 10 - metabolism; Chaperonin 60 - metabolism; Chaperonins - metabolism; DM-MBP; double mutant of maltose binding protein; GroEL; GroES; Humans; Kinetics; Light; MBP; Mutant Proteins - chemistry; Myelin Basic Protein - chemistry; Myelin Basic Protein - genetics; Protein Folding; Scattering, Radiation; Solutions - metabolism; Aggregation; MBP; DM-MBP; GroES; GroEL
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2011.05.031

► Does the GroEL/GroES cavity accelerate a folding reaction? ► Rate of refolding DM-MBP with GroEL/GroES is faster than in free solution. ► Concentration-dependent refolding and light scattering suggest reversible aggregation. ► This suggested relative slowing in solution via reversible aggregation. ► When aggregation was prevented, concentration dependence was abolished. ► The folding rate in solution became equal to the rate of folding inside GroEL/GroES. Under “permissive” conditions at 25°C, the chaperonin substrate protein DM-MBP refolds 5–10 times more rapidly in the GroEL/GroES folding chamber than in free solution. This has been suggested to indicate that the chaperonin accelerates polypeptide folding by entropic effects of close confinement. Here, using native-purified DM-MBP, we show that the different rates of refolding are due to reversible aggregation of DM-MBP while folding free in solution, slowing its kinetics of renaturation: the protein exhibited concentration-dependent refolding in solution, with aggregation directly observed by dynamic light scattering. When refolded in chloride-free buffer, however, dynamic light scattering was eliminated, refolding became concentration-independent, and the rate of refolding became the same as that in GroEL/GroES. The GroEL/GroES chamber thus appears to function passively toward DM-MBP.