ATP-Independent Mechanism of Chaperone-Mediated Protein Folding

Abstract

The folding of proteins into their native structure is crucial for the functioning of all biological processes. Molecular chaperones are guardians of the proteome that assist in protein folding and act to prevent the accumulation of aberrant protein conformations that can lead to proteotoxicity. Despite their important functions in protein folding homeostasis, it is not entirely clear the underlying mechanisms of how chaperones assist in protein folding. This thesis focused on understanding the mechanism of the specific class of chaperones that do not use ATP or other cofactors to regulate their chaperone activity. These ATP-independent chaperones are generally assumed to act as “holdase” chaperones that work by tightly holding onto non-native proteins and thus preventing them from aggregating. This traditional “holdase” perspective that ATP-independent chaperones are not directly involved in the folding process had been recently challenged. Our recent studies using the periplasmic chaperone Spy illustrates that client proteins can explore different conformational states while bound to this ATP-independent chaperone. This thesis aims to (1) understand the principles underlying how proteins can fold while they remain continuously bound to a chaperone, and (2) examine whether this folding-while-bound mechanism is utilized by other classic “holdase” chaperones. In the first part of this thesis, super Spy variants (H96L and Q100L) were used to show that strengthening the interaction between Spy and its client protein strongly inhibits the ability of the client to fold while chaperone bound. The results showed that more tightly Spy binds to its clients, the more it slows the folding rate of the bound client. Efficient chaperone-mediated folding while bound appears to represent an evolutionary balance between interactions of sufficient strength to mediate folding and interactions that are too tight, which tend to inhibit protein folding. In the second part of this thesis, trigger factor was used as another ATP-independent chaperone to test if allowing proteins to explore different conformational states while chaperone bound could be an underlying principle that also applies to other chaperones that have been thought to act as simple “holdases”. Trigger factor rapidly binds to partially folded glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and prevents it from non-productive self-association by shielding oligomeric interfaces from aggregation, consistent with a role as an aggregation inhibiting holdase chaperone. Surprisingly, non-native GAPDH folds into a monomeric but otherwise rather native-like intermediate state while trigger factor-bound. Upon release from trigger factor, this mostly folded monomeric GAPDH rapidly self-associates and acquires enzymatic activity. The work presented here shows how chaperones can bridge the holdase-foldase divide and demonstrates how an ATP-independent chaperone can be actively involved in the protein folding process.