High affinity of Skp to OmpC revealed by single-molecule detection

Abstract

AbstractOuter membrane proteins (OMPs) are essential to Gram-negative bacteria, and they need molecular chaperones to prevent from aggregation in periplasm during the OMPs biogenesis. Seventeen kilodalton protein (Skp) is the major protein for this purpose. Here we used singlemolecule detection (SMD) to study the stoichiometry modulation of Skp in binding with outer membrane protein C (OmpC) from Escherichia coli. To accomplish our task, we developed the tool of portion selectively chosen fluorescence correlation spectroscopy (pscFCS). We found that Skp binds OmpC with high affinity. The half concentration for Skp to form homotrimer Skp3 (C1/2) was measured to be 250 nM. Under the Skp concentrations far below C1/2 OmpC can recruit Skp monomers to form OmpC·Skp3. The affinity of the process is in picomolar range, indicating that the trimerization of Skp in OmpC·Skp3 complex is induced by OmpC-Skp interaction even though free Skp3 is rarely present. In the concentration range that Skp3 is the predominant form, OmpC may directly interact with Skp3. Under micro-molar concentrations of Skp, the formation of OmpC·(Skp3)2 was observed. Our results suggest that the fine-tuned modulation of Skp composition stoichiometry plays an important role in the safe-guarding and quality control mechanism of OMPs in the periplasm.