Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism

2011 ◽  
Vol 438 (3) ◽  
pp. 505-511 ◽  
Author(s):  
Si Wu ◽  
Xi Ge ◽  
Zhixin Lv ◽  
Zeyong Zhi ◽  
Zengyi Chang ◽  
...  
Keyword(s):  
Quality Control ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Single Molecule ◽  
Outer Membrane Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Aqueous Environment ◽  
Periplasmic Space ◽  
Control Factors

The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone–substrate interaction may be essential for the quality control of the biogenesis of OMPs

scholarly journals Molecular mechanism of networking among DegP, Skp and SurA in periplasm for biogenesis of outer membrane proteins

2020 ◽  
Vol 477 (16) ◽  
pp. 2949-2965
Author(s):  
Chen Yang ◽  
Sijia Peng ◽  
Chunlai Chen ◽  
Xin Sheng Zhao
Keyword(s):  
Quality Control ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Molecular Mechanism ◽  
Single Molecule ◽  
Ternary Complex ◽  
Outer Membrane Proteins ◽  
The Self ◽  
Control Factors ◽  
Comprehensive Picture

The biogenesis of outer membrane proteins (OMPs) is an extremely challenging process. In the periplasm of Escherichia coli, a group of quality control factors work together to exercise the safe-guard and quality control of OMPs. DegP, Skp and SurA are the three most prominent ones. Although extensive investigations have been carried out, the molecular mechanism regarding the networking among these proteins remains mostly mysterious. Our group has previously studied the molecular interactions of OMPs with SurA and Skp, using single-molecule detection (SMD). In this work, again using SMD, we studied how OmpC, a representative of OMPs, interacts with DegP, Skp and SurA collectively. Several important discoveries were made. The self-oligomerization of DegP to form hexamer occurs over hundred micromolars. When OmpC is in a monomer state at a low concentration, the OmpC·DegP6 and OmpC·DegP24 complexes form when the DegP concentration is around sub-micromolars and a hundred micromolars, respectively. High OmpC concentration promotes the binding affinity of DegP to OmpC by ∼100 folds. Skp and SurA behave differently when they interact synergistically with DegP in the presence of substrate. DegP can degrade SurA-protected OmpC, but Skp-protected OmpC forms the ternary complex OmpC·(Skp3)n·DegP6 (n = 1,2) to resist the DegP-mediated degradation. Combined with previous results, we were able to depict a comprehensive picture regarding the molecular mechanism of the networking among DegP, Skp and SurA in the periplasm for the OMPs biogenesis under physiological and stressed conditions.

scholarly journals Single Molecule Localization Microscopy (SMLM) imaging of bacteria: A sample preparation guide

2020 ◽  
Author(s):  
Sachith D. Gunasinghe ◽  
Kirstin D. Elgass ◽  
Toby D. M. Bell ◽  
Trevor Lithgow
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Single Molecule ◽  
Spatial Organization ◽  
Outer Membrane Proteins ◽  
Fluorescent Microscopy ◽  
Super Resolution ◽  
Model Systems ◽  
Invaluable Tool ◽  
Optical Reconstruction

Abstract In recent years Super-resolution microscopy has become an invaluable tool to noninvasively interrogate the membrane architecture of bacteria to study the spatial organization of proteins associated with membranes, which in turn help us to understand how bacteria have evolved to exploit environmental niches. Model systems like Escherichia coli and Caulobacter cresentus have been used to study the spatiotemporal organization of membrane proteins. Like most gram-negative bacteria, the outer membrane of E.coli is populated with β-barrel proteins, which serve as selective channels where exchange of small molecules take place. Surface exposed domains in these channels provide means to fluorescently label and utilise them for fluorescent microscopy studies to investigate their spatial organization at the outer membrane. Here, we describe a methodology to fluorescently label outer membrane proteins in E.coli and study their spatial organization using direct stochastic optical reconstruction microscopy (dSTORM).

scholarly journals Affinity of Skp to OmpC revealed by single-molecule detection

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sichen Pan ◽  
Chen Yang ◽  
Xin Sheng Zhao
Keyword(s):  
Single Molecule ◽  
Molecular Chaperones ◽  
Outer Membrane Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Hill Coefficient ◽  
Stoichiometric Ratio ◽  
Single Molecule Level

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.

Periplasmic quality control in biogenesis of outer membrane proteins

2015 ◽  
Vol 43 (2) ◽  
pp. 133-138 ◽  
Author(s):  
Zhi Xin Lyu ◽  
Xin Sheng Zhao
Keyword(s):  
Quality Control ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Diverse Range ◽  
Multifunctional Protein ◽  
Proposed Model ◽  
Periplasmic Proteins ◽  
Basic Functions ◽  
Biochemical Analyses

The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.

scholarly journals Lateral gate dynamics of the bacterial translocon during cotranslational membrane protein insertion

2021 ◽  
Vol 118 (26) ◽  
pp. e2100474118
Author(s):  
Evan Mercier ◽  
Xiaolin Wang ◽  
Manisankar Maiti ◽  
Wolfgang Wintermeyer ◽  
Marina V. Rodnina
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Biological Significance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Phospholipid Bilayer ◽  
Membrane Phospholipids ◽  
Model Free ◽  
Lateral Gate

During synthesis of membrane proteins, transmembrane segments (TMs) of nascent proteins emerging from the ribosome are inserted into the central pore of the translocon (SecYEG in bacteria) and access the phospholipid bilayer through the open lateral gate formed of two helices of SecY. Here we use single-molecule fluorescence resonance energy transfer to monitor lateral-gate fluctuations in SecYEG embedded in nanodiscs containing native membrane phospholipids. We find the lateral gate to be highly dynamic, sampling the whole range of conformations between open and closed even in the absence of ligands, and we suggest a statistical model-free approach to evaluate the ensemble dynamics. Lateral gate fluctuations take place on both short (submillisecond) and long (subsecond) timescales. Ribosome binding and TM insertion do not halt fluctuations but tend to increase sampling of the open state. When YidC, a constituent of the holotranslocon, is bound to SecYEG, TM insertion facilitates substantial opening of the gate, which may aid in the folding of YidC-dependent polytopic membrane proteins. Mutations in lateral gate residues showing in vivo phenotypes change the range of favored states, underscoring the biological significance of lateral gate fluctuations. The results suggest how rapid fluctuations of the lateral gate contribute to the biogenesis of inner-membrane proteins.

scholarly journals Fast slow folding of an Outer Membrane Porin

2021 ◽  
Author(s):  
Eve E. Weatherill ◽  
Monifa A. Fahie ◽  
David P. Marshall ◽  
Rachel A. Andvig ◽  
Matthew R. Cheetham ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Globular Proteins ◽  
Protein G ◽  
Outer Membrane Porin ◽  
Unfolded State

AbstractIn comparison to globular proteins, the spontaneous folding and insertion of β-barrel membrane proteins is surprisingly slow, typically occurring on the order of minutes. Using single-molecule Förster Resonance Energy Transfer to report on the folding of fluorescently-labelled Outer Membrane Protein G we measured the real-time insertion of a β-barrel membrane protein from an unfolded state. Folding events were rare, and fast (<20 ms); occurring immediately upon arrival at the membrane. This combination of infrequent, but rare, folding resolves this apparent dichotomy between slow ensemble kinetics, and the typical timescales of biomolecular folding.

scholarly journals Chaperone Spy Protects Outer Membrane Proteins from Folding Stress via Dynamic Complex Formation

mBio ◽  
2021 ◽  
Author(s):  
Wei He ◽  
Gangjin Yu ◽  
Tianpeng Li ◽  
Ling Bai ◽  
Yuanyuan Yang ◽  
...  
Keyword(s):  
Quality Control ◽  
Membrane Proteins ◽  
Complex Formation ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Control Network ◽  
Comprehensive Understanding ◽  
Antibiotic Development ◽  
Bacterial Pathogenicity

Outer membrane proteins (OMPs) play critical roles in bacterial pathogenicity and provide a new niche for antibiotic development. A comprehensive understanding of the OMP quality control network will strongly impact antimicrobial discovery.

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