Native-like structure of a protein-folding intermediate bound to the chaperonin GroEL.
The chaperonin GroEL binds nonnative proteins in its central channel through hydrophobic interactions and initiates productive folding in this space underneath bound co-chaperone, GroES, in the presence of ATP. The questions of where along the folding pathway a protein is recognized by GroEL, and how much structure is present in a bound substrate have remained subjects of discussion, with some experiments suggesting that bound forms are fully unfolded and others suggesting that bound species are partially structured. Here we have studied a substrate protein, human dihydrofolate reductase (DHFR), observing in stopped-flow fluorescence experiments that it can rapidly bind to GroEL at various stages of folding. We have also analyzed the structure of the GroEL-bound protein using hydrogen-deuterium exchange and NMR spectroscopy. The pattern and magnitude of amide proton protection indicate that the central parallel beta-sheet found in native DHFR is present in a moderately stable state in GroEL-bound DHFR. Considering that the strands are derived from distant parts of the primary structure, this suggests that a native-like global topology is also present. We conclude that significant native-like structure is present in protein-folding intermediates bound to GroEL.
Goldberg
M S
MS
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510, USA.
Zhang
J
J
Sondek
S
S
Matthews
C R
CR
Fox
R O
RO
Horwich
A L
AL
D000577:Amides / Q000737:chemistry; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D017022:Flow Injection Analysis / Q000379:methods; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*
D000577:Amides; D018834:Chaperonin 60; D011522:Protons; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase