Allostery in GroEL: Its Role in the Refolding of Protein Substrates

Abstract

The Escherichia coli chaperonin GroEL assists in the re-folding of misfolded substrate proteins (SPs). In response to the binding of ATP, GroEL undergoes large, allosteric structural transitions, resulting in an expansion of its central cavity and a capping of the cavity by the co-chaperonin GroES. Bound SP is released into the central cavity following the structural transitions. The exact mechanism by which GroEL assists in the re-folding of SPs is unknown, though there is evidence that GroEL has the ability to forcefully unfold bound SPs, giving them another chance to fold to the native state.

The studies in this dissertation concentrate on relating the allosteric domain movements of GroEL to the unfolding of SPs: 1) As a means of controlling the domain movements, an intersubunit salt bridge was replaced with a pair of cysteine residues, allowing for the controlled introduction of cross-links that could tether the GroEL rings in their closed conformation. 2) The possible allosteric basis of SP's ability to stimulate the ATPase activity of GroEL was explored using standard kinetic assays. 3) The kinetics of GroES release from the GroEL/GroES complex in response to ATP binding were studied using stopped-flow fluorescence measurements, with an emphasis on determining why SP binding accelerates the rate of release. From these studies, it was concluded that the subunits within a GroEL ring move in a single concerted motion, maximizing the potential unfolding force exerted by GroEL against bound SP. It was also found that SP stimulates ATPase activity by binding to and holding a ring in the more active, closed conformation. To do this, SP must exert a force on the ring, and in order to undergo its structural changes, GroEL must in turn perform work on the SP. GroES release is stimulated for a similar reason. Since unfolded SP increases the number of reaction cycles and decreases the amount of time SP spends encapsulated in the central cavity, it is proposed that a GroEL-assisted refolding mechanism that includes an active unfolding event makes the most sense in physiological terms.