| Abstract: |
Outer membrane protein (OMP) biogenesis in gram-negative bacteria is one of many processes mediated in part by molecular chaperones. A network of periplasmic chaperones that includes SurA, Skp, and FkpA binds unfolded OMPs (uOMPs) in dynamic conformational ensembles to suppress uOMP aggregation and promote proper OMP folding and function. FkpA is a dimeric chaperone primarily involved in suppressing uOMP aggregation in response to heat-shock stress, but its mechanism of action is understudied compared to other periplasmic chaperones. To probe FkpA chaperone function, we assayed the folding of a model uOMP and found that unlike other periplasmic chaperones, FkpA improves the overall folding efficiency but halves folding rate for the uOMP. To further investigate the interaction between this chaperone and the uOMP client, we heavily utilized sedimentation velocity analytical ultracentrifugation (SV-AUC) titration experiments. Titrations were globally fit to determine an FkpA-uOMP binding affinity intermediate between the known affinities of Skp and SurA for uOMPs. The binding shows a large dependence on urea concentration, suggesting an extensive binding interface. Indeed, initial characterizations of the binding interface using photo-crosslinking indicates that it spans the inner surfaces of both the N-terminal dimerization domain and the C-terminal peptidyl-prolyl isomerization domain of FkpA. Together these results imply that FkpA’s distinct effect on uOMP folding reflects its intermediate binding affinity and that FkpA dimers achieve this function by utilizing an extensive chaperone-client interface that spans both the N- and C- domains. This work also demonstrates the usefulness of sedimentation velocity experiments for investigating interactions between chaperones and their unfolded clients. |
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