Single molecule microscopy reveals diverse actions of substrate sequences that impair ClpX AAA+ ATPase function.

ABSTRACT: AAA+ (ATPases Associated with diverse cellular Activities) proteases unfold substrate proteins by pulling the substrate polypeptide through a narrow pore. To overcome the barrier to unfolding, substrates may require extended association with the ATPase. Failed unfolding attempts can lead to a slip of grip, which may result in substrate dissociation, but how substrate sequence affects slippage is unresolved. Here, we measured single molecule dwell time using total internal reflection fluorescence microscopy, scoring time-dependent dissociation of engaged substrates from bacterial AAA+ ATPase unfoldase/translocase ClpX. Substrates comprising a stable domain resistant to unfolding and a C-terminal unstructured tail, tagged with a degron for initiating translocase insertion, were used to deter

SUBMITTER: Wang X 

PROVIDER: S-EPMC9531181 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature