Mizellen, Bizellen und Nanoscheiben: Einfluss von membranimitierenden Umgebungen auf die Membranproteindynamik

Frey, Lukas; Lakomek, Nils‐Alexander; Riek, Roland; Bibow, Stefan

doi:10.1002/ange.201608246

Cited by 7 publications

(2 citation statements)

References 42 publications

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“…Recent studies, for example, have shown for one β-barrel protein (OmpX) that while it forms a stable β-barrel in detergent micelles, bicelles, and nanodiscs, the dynamics of the protein differ substantially between the detergent and lipid environments. 29 Furthermore, in vivo the N-terminal region of LptD, where binding to the antibiotic should occur, is bound to LptA as part of the Lpt transport macromolecular complex. The absence of LptA in vitro may also affect the structure or dynamics of this region in LptD.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

A Peptidomimetic Antibiotic Interacts with the Periplasmic Domain of LptD from Pseudomonas aeruginosa

et al. 2018

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The outer membrane (OM) in Gram-negative bacteria is an asymmetric bilayer with mostly lipopolysaccharide (LPS) molecules in the outer leaflet. During OM biogenesis, new LPS molecules are transported from their site of assembly on the inner membrane to the OM by seven LPS transport proteins (LptA-G). The complex formed between the integral β-barrel OM protein LptD and the lipoprotein LptE is responsible for transporting LPS from the periplasmic side of the OM to its final location on the cell surface. Because of its essential function in many Gram-negative bacteria, the LPS transport pathway is an interesting target for the development of new antibiotics. A family of macrocyclic peptidomimetics was discovered recently that target LptD and inhibit LPS transport specifically in Pseudomonas spp. The related molecule Murepavadin is in clinical development for the treatment of life-threatening infections caused by P. aeruginosa. To characterize the interaction of these antibiotics with LptD from P. aeruginosa, we characterized the binding site by cross-linking to a photolabeling probe. We used a hypothesis-free mass spectrometry-based proteomic approach to provide evidence that the antibiotic cross-links to the periplasmic segment of LptD, containing a β-jellyroll domain and an N-terminal insert domain characteristic of Pseudomonas spp. Binding of the antibiotic to the periplasmic segment is expected to block LPS transport, consistent with the proposed mode of action and observed specificity of these antibiotics. These insights may prove valuable for the discovery of new antibiotics targeting the LPS transport pathway in other Gram-negative bacteria.

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Section: ■ Results and Discussionmentioning

confidence: 99%

A Peptidomimetic Antibiotic Interacts with the Periplasmic Domain of LptD from Pseudomonas aeruginosa

et al. 2018

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“…22−24 Bicelles and nanodiscs can restore the functional activity of proteins characterized in micellar systems. 25,26 Nanodiscs have reasonable tumbling times for solution state NMR, but they require the purification of MSPs, which themselves present an additional protein into the system capable of producing unpredictable interactions with the protein of interest. Numerous solid-state NMR (SSNMR) protein structures circumvent curvature issues with extruded or freeze−thawed vesicles, yet these studies still typically use vesicles with non-native compositions of lipids.…”

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confidence: 99%

Structure and Dynamics of Membrane Proteins and Membrane Associated Proteins with Native Bicelles from Eukaryotic Tissues

et al. 2017

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In vitro studies of protein structure, function, and dynamics typically preclude the complex range of molecular interactions found in living tissues. In vivo studies elucidate these complex relationships, yet they are typically incompatible with the extensive and controlled biophysical experiments available in vitro. We present an alternative approach by extracting membranes from eukaryotic tissues to produce native bicelles to capture the rich and complex molecular environment of in vivo studies while retaining the advantages of in vitro experiments. Native bicelles derived from chicken egg or mouse cerebrum tissues contain a rich composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), lysolipids, cholesterol, ceramides (CM), and sphingomyelin (SM). The bicelles also contain source-specific lipids such as triacylglycerides (TAGs) and sulfatides from egg and brain tissues, respectively. With the influenza hemagglutinin fusion peptide (HAfp) and the C-terminal Src homology domain of lymphocyte-specific protein-tyrosine kinase (lck-cSH2), we show that membrane proteins and membrane associated proteins reconstituted in native bicelles produce high-resolution NMR data and probe native protein-lipid interactions.

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Membrane Proteins Have Distinct Fast Internal Motion and Residual Conformational Entropy

et al. 2020

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The internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side‐chain dynamics of the α‐helical sensory rhodopsin II and the β‐barrel outer membrane protein W have been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Despite their differing topologies, both proteins have a similar distribution of methyl‐bearing side‐chain motion that is largely independent of membrane mimetic. The methyl‐bearing side chains of both proteins are, on average, more dynamic in the ps–ns timescale than any soluble protein characterized to date. Accordingly, both proteins retain an extraordinary residual conformational entropy in the folded state, which provides a counterbalance to the absence of the hydrophobic effect. Furthermore, the high conformational entropy could greatly influence the thermodynamics underlying membrane‐protein functions, including ligand binding, allostery, and signaling.

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Mizellen, Bizellen und Nanoscheiben: Einfluss von membranimitierenden Umgebungen auf die Membranproteindynamik

Cited by 7 publications

References 42 publications

A Peptidomimetic Antibiotic Interacts with the Periplasmic Domain of LptD from Pseudomonas aeruginosa

A Peptidomimetic Antibiotic Interacts with the Periplasmic Domain of LptD from Pseudomonas aeruginosa

Structure and Dynamics of Membrane Proteins and Membrane Associated Proteins with Native Bicelles from Eukaryotic Tissues

Membrane Proteins Have Distinct Fast Internal Motion and Residual Conformational Entropy

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