Free and Chaperone Bound Unfolded States of Outer Membrane Proteins

Abstract

Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with chaperones. In the case of bacterial outer-membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance, because they remain unfolded for extended periods of time during their biogenesis and rely on interactions with periplasmic chaperones to prevent aggregation and support correct folding. Here, we study the unfolded-state properties of outer-membrane proteins. Using a combination of ensemble and single-molecule spectroscopy techniques including single-molecule FRET, we find that under strongly denaturing conditions and in the absence of chaperones, OMPs adopt conformationally heterogeneous unfolded states that lack the fast chain reconfiguration motions expected for an unstructured, fully unfolded chain. Interestingly, when complexed with periplasmic chaperones seventeen kilodalton protein (Skp) and survival factor A (SurA), we observe for the protein OmpLA adopts an expanded, yet squished conformational ensemble, in which the overall broad distribution of unfolded states is preserved. Differently sized OMPs like OmpX (8 β-strands) and OmpF (16 β-strands) allow a broader perspective on chaperone enabled conformations of OMPs. These findings indicate that periplasmic chaperones Skp and SurA prestructure OMPs for their next binding partner (e.g., the β-barrel assembly machinery (BAM)) to facilitate insertion and proper folding into the outer membrane.