Related Experiment Video
Updated: Dec 9, 2025
Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization
Published on: February 27, 2020
Affinity of Skp to OmpC revealed by single-molecule detection
Sichen Pan1,2, Chen Yang1,2, Xin Sheng Zhao3,4
1Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, and Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China.
Abstract:
Outer membrane proteins (OMPs) are essential to gram-negative bacteria, and molecular chaperones prevent the OMPs from aggregation in the periplasm during the OMPs biogenesis. Skp is one of the molecular chaperones for this purpose. Here, we combined single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy to study the affinity and stoichiometric ratio of Skp in its binding with OmpC at the single-molecule level. The half concentration of the Skp self-trimerization (C1/2) was measured to be (2.5 ± 0.7) × 102 nM. Under an Skp concentration far below the C1/2, OmpC could recruit Skp monomers to form OmpC·Skp3. The affinity to form the OmpC·Skp3 complex was determined to be (5.5 ± 0.4) × 102 pM with a Hill coefficient of 1.6 ± 0.2. Under the micromolar concentrations of Skp, the formation of OmpC·(Skp3)2 was confirmed, and the dissociation constant of OmpC·(Skp3)2 was determined to be 1.2 ± 0.4 μM. The precise information will help us to quantitatively depict the role of Skp in the biogenesis of OMPs.
