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.
Highlights
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
Since a single OMP polypeptide is synthesized in the cytoplasm in an unfolded form, they have to be transported through the aqueous periplasm and to be folded in the outer membrane (OM), several periplasmic quality control factors, such as SurA, Skp and DegP, are involved in the protection of OMPs from mis-folding and aggregation[2,3,4,5]
To study a specific subpopulation, we developed portion-selectively-chosen fluorescence correlation spectroscopy (pscFCS) which used both single-molecule fluorescence resonance energy transfer (smFRET) and fluorescence correlation spectroscopy (FCS)
Summary
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. 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. OMPs are in the equilibrium between the chaperone-unbound state and the chaperone-bound s tate[18], and Skp is in the equilibrium between the Skp monomer and the S kp3 trimer[16] These equilibria will blur the information at the ensemble level. It is unclear what is exactly the stoichiometric ratio in the OMP-Skp complex when the Skp and OMP monomers are predominant in the solution. We picked out a portion of the smFRET components and carried out an FCS study on the molecular weight of the Scientific Reports | (2020) 10:14871
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