Interaction of GroEL with a highly structured folding intermediate: iterative binding cycles do not involve unfolding.
The GroE proteins are molecular chaperones involved in protein folding. The general mechanism by which they facilitate folding is still enigmatic. One of the central open questions is the conformation of the GroEL-bound nonnative protein. Several suggestions have been made concerning the folding stage at which a protein can interact with GroEL. Furthermore, the possibility exists that binding of the nonnative protein to GroEL results in its unfolding. We have addressed these issues that are basic for understanding the GroE-mediated folding cycle by using folding intermediates of an Fab antibody fragment as molecular probes to define the binding properties of GroEL. We show that, in addition to binding to an early folding intermediate, GroEL is able to recognize and interact with a late quaternary-structured folding intermediate (Dc) without measurably unfolding it. Thus, the prerequisite for binding is not a certain folding stage of a nonnative protein. In contrast, general surface properties of nonnative proteins seem to be crucial for binding. Furthermore, unfolding of a highly structured intermediate does not necessarily occur upon binding to GroEL. Folding of Dc in the presence of GroEL and ATP involves cycles of binding and release. Because in this system no off-pathway reactions or kinetic traps are involved, a quantitative analysis of the reactivation kinetics observed is possible. Our results indicate that the association reaction of Dc and GroEL in the presence of ATP is rather slow, whereas in the absence of ATP association is several orders of magnitude more efficient. Therefore, it seems that ATP functions by inhibiting reassociation rather than promoting release of the bound substrate.
Lilie
H
H
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Buchner
J
J
D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D018834:Chaperonin 60; D007140:Immunoglobulin Fab Fragments; D046911:Macromolecular Substances