| 1. |
- Castegnaro, Filippo, 1994, et al.
(författare)
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Preparation of Protein-Enriched Outer Membrane Vesicles from Escherichia Coli for In Situ Structural Biology of Outer Membrane Proteins
- 2023
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Ingår i: Methods in molecular biology (Clifton, N.J.). - 1940-6029. ; , s. 247-257
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Bokkapitel (refereegranskat)abstract
- Bacterial outer membrane vesicles (OMVs) can be selectively enriched with one or more outer membrane proteins to allow the biophysical characterization of these membrane proteins embedded in the native cellular environment. Unlike reconstituted artificial membrane environments, OMVs maintain the native lipid composition as well as the lipid asymmetry of bacterial outer membranes. Here, we describe in detail the steps necessary to prepare OMVs, which contain high levels of a designated protein of interest, and which are of sufficient homogeneity and purity to perform biophysical characterizations using high-resolution methods such as atomic force microscopy, electron microscopy, or single-molecule force spectroscopy.
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| 2. |
- Darius, Šulskis, et al.
(författare)
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Structural basis of DegP protease temperature-dependent activation
- 2021
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 7:50
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Tidskriftsartikel (refereegranskat)abstract
- Protein quality control is an essential cellular function mainly executed by a vast array of different proteases and molecular chaperones. One of the bacterial high temperature requirement A (HtrA) protein family members, the homo-oligomeric DegP protease, plays a crucial role in the Escherichia coli protein quality control machinery by removing unfolded proteins or preventing their aggregation and chaperoning them to their final folded state within the periplasm. DegP contains two regulatory PDZ domains, which play key roles in substrate recognition and in the transformation of DegP between inactive hexameric and proteolytic active cage-like structures. Here, we analyze the interaction and dynamics of the DegP PDZ domains underlying this transformation by high-resolution NMR spectroscopy complemented with biochemical cleavage assays. We identify an interdomain molecular lock, which controls the interactions between the two PDZ domains, regulated by fine-tuned temperature-dependent protein dynamics, and which is potentially conserved in proteins harboring tandem PDZ domains.
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| 3. |
- Lindström, Michelle, et al.
(författare)
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Lsm7 phase-separated condensates trigger stress granule formation
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Stress granules are non-membranous organelles connected to stress responses and age-related disease. Here, the authors identify a conserved yeast protein, Lsm7, that facilitates stress granule formation through dynamic liquid-liquid phase separation condensates upon 2-deoxy-D-glucose-induced stress. Stress granules (SGs) are non-membranous organelles facilitating stress responses and linking the pathology of age-related diseases. In a genome-wide imaging-based phenomic screen, we identify Pab1 co-localizing proteins under 2-deoxy-D-glucose (2-DG) induced stress in Saccharomyces cerevisiae. We find that deletion of one of the Pab1 co-localizing proteins, Lsm7, leads to a significant decrease in SG formation. Under 2-DG stress, Lsm7 rapidly forms foci that assist in SG formation. The Lsm7 foci form via liquid-liquid phase separation, and the intrinsically disordered region and the hydrophobic clusters within the Lsm7 sequence are the internal driving forces in promoting Lsm7 phase separation. The dynamic Lsm7 phase-separated condensates appear to work as seeding scaffolds, promoting Pab1 demixing and subsequent SG initiation, seemingly mediated by RNA interactions. The SG initiation mechanism, via Lsm7 phase separation, identified in this work provides valuable clues for understanding the mechanisms underlying SG formation and SG-associated human diseases.
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| 4. |
- Manioglu, S., et al.
(författare)
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Antibiotic polymyxin arranges lipopolysaccharide into crystalline structures to solidify the bacterial membrane
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Polymyxins are last-resort antibiotics with potent activity against multi-drug resistant pathogens. They interact with lipopolysaccharide (LPS) in bacterial membranes, but mechanistic details at the molecular level remain unclear. Here, we characterize the interaction of polymyxins with native, LPS-containing outer membrane patches of Escherichia coli by high-resolution atomic force microscopy imaging, along with structural and biochemical assays. We find that polymyxins arrange LPS into hexagonal assemblies to form crystalline structures. Formation of the crystalline structures is correlated with the antibiotic activity, and absent in polymyxin-resistant strains. Crystal lattice parameters alter with variations of the LPS and polymyxin molecules. Quantitative measurements show that the crystalline structures decrease membrane thickness and increase membrane area as well as stiffness. Together, these findings suggest the formation of rigid LPS-polymyxin crystals and subsequent membrane disruption as the mechanism of polymyxin action and provide a benchmark for optimization and de novo design of LPS-targeting antimicrobials. Manioglu et al use high-resolution atomic force microscopy to resolve how polymyxins interact with the bacterial membrane. Polymyxins arrange the bacterial lipids into regular hexagonal structures that stiffen the membrane and lead to rupture.
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| 5. |
- Mas, G., et al.
(författare)
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The Periplasmic Chaperones Skp and SurA
- 2019
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Ingår i: Bacterial Cell Walls and Membranes. - Cham : Springer International Publishing. - 0306-0225.
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The periplasm of Gram-negative bacteria contains a specialized chaperone network that facilitates the transport of unfolded membrane proteins to the outer membrane as its primary functional role. The network, involving the chaperones Skp and SurA as key players and potentially additional chaperones, is indispensable for the survival of the cell. Structural descriptions of the apo forms of these molecular chaperones were initially provided by X-ray crystallography. Subsequently, a combination of experimental biophysical methods including solution NMR spectroscopy provided a detailed understanding of full-length chaperone–client complexes. The data showed that conformational changes and dynamic re-organization of the chaperones upon client binding, as well as client dynamics on the chaperone surface are crucial for function. This chapter gives an overview of the structure-function relationship of the dynamic conformational rearrangements that regulate the functional cycles of the periplasmic molecular chaperones Skp and SurA. © 2019, Springer Nature Switzerland AG.
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| 6. |
- Palica, Katarzyna, 1992-, et al.
(författare)
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α-Aminophosphonate inhibitors of metallo-β-lactamases NDM-1 and VIM-2
- 2023
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Ingår i: RSC Medicinal Chemistry. - : Royal Society of Chemistry. - 2632-8682. ; 14:11, s. 2277-2300
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Tidskriftsartikel (refereegranskat)abstract
- The upswing of antibiotic resistance is an escalating threat to human health. Resistance mediated by bacterial metallo-β-lactamases is of particular concern as these enzymes degrade β-lactams, our most frequently prescribed class of antibiotics. Inhibition of metallo-β-lactamases could allow the continued use of existing β-lactam antibiotics, such as penicillins, cephalosporins and carbapenems, whose applicability is becoming ever more limited. The design, synthesis, and NDM-1, VIM-2, and GIM-1 inhibitory activities (IC50 4.1–506 μM) of a series of novel non-cytotoxic α-aminophosphonate-based inhibitor candidates are presented herein. We disclose the solution NMR spectroscopic and computational investigation of their NDM-1 and VIM-2 binding sites and binding modes. Whereas the binding modes of the inhibitors are similar, VIM-2 showed a somewhat higher conformational flexibility, and complexed a larger number of inhibitor candidates in more varying binding modes than NDM-1. Phosphonate-type inhibitors may be potential candidates for development into therapeutics to combat metallo-β-lactamase resistant bacteria.
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| 7. |
- Ritzmann, N., et al.
(författare)
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Mechanical Unfolding and Refolding of Single Membrane Proteins by Atomic Force Microscopy
- 2020
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Ingår i: Expression, Purification, and Structural Biology of Membrane Proteins. - New York, NY : Springer US. - 1064-3745. ; , s. 359-372
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Atomic force microscopy (AFM)-based single-molecule force spectroscopy allows direct physical manipulation of single membrane proteins under near-physiological conditions. It can be applied to study mechanical properties and molecular interactions as well as unfolding and folding pathways of membrane proteins. Here, we describe the basic procedure to study membrane proteins by single-molecule force spectroscopy and discuss general requirements of the experimental setup as well as common pitfalls typically encountered when working with membrane proteins in AFM. © 2020, Springer Science+Business Media, LLC, part of Springer Nature.
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| 8. |
- Thoma, Johannes, 1985, et al.
(författare)
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Architects of their own environment: How membrane proteins shape the Gram-negative cell envelope.
- 2022
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Ingår i: Advances in protein chemistry and structural biology. - : Elsevier. - 1876-1631. ; 128, s. 1-34
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Forskningsöversikt (refereegranskat)abstract
- Gram-negative bacteria are surrounded by a complex multilayered cell envelope, consisting of an inner and an outer membrane, and separated by the aqueous periplasm, which contains a thin peptidoglycan cell wall. These bacteria employ an arsenal of highly specialized membrane protein machineries to ensure the correct assembly and maintenance of the membranes forming the cell envelope. Here, we review the diverse protein systems, which perform these functions in Escherichia coli, such as the folding and insertion of membrane proteins, the transport of lipoproteins and lipopolysaccharide within the cell envelope, the targeting of phospholipids, and the regulation of mistargeted envelope components. Some of these protein machineries have been known for a long time, yet still hold surprises. Others have only recently been described and some are still missing pieces or yet remain to be discovered.
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| 9. |
- Thoma, Johannes, 1985, et al.
(författare)
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Fake It 'Till You Make It-The Pursuit of Suitable Membrane Mimetics for Membrane Protein Biophysics
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins evolved to reside in the hydrophobic lipid bilayers of cellular membranes. Therefore, membrane proteins bridge the different aqueous compartments separated by the membrane, and furthermore, dynamically interact with their surrounding lipid environment. The latter not only stabilizes membrane proteins, but directly impacts their folding, structure and function. In order to be characterized with biophysical and structural biological methods, membrane proteins are typically extracted and subsequently purified from their native lipid environment. This approach requires that lipid membranes are replaced by suitable surrogates, which ideally closely mimic the native bilayer, in order to maintain the membrane proteins structural and functional integrity. In this review, we survey the currently available membrane mimetic environments ranging from detergent micelles to bicelles, nanodiscs, lipidic-cubic phase (LCP), liposomes, and polymersomes. We discuss their respective advantages and disadvantages as well as their suitability for downstream biophysical and structural characterization. Finally, we take a look at ongoing methodological developments, which aim for direct in-situ characterization of membrane proteins within native membranes instead of relying on membrane mimetics.
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| 10. |
- Thoma, Johannes, 1985, et al.
(författare)
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High-Resolution In Situ NMR Spectroscopy of Bacterial Envelope Proteins in Outer Membrane Vesicles
- 2020
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 59:17, s. 1656-1660
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Tidskriftsartikel (refereegranskat)abstract
- The cell envelope of Gram-negative bacteria is an elaborate cellular environment, consisting of two lipid membranes separated by the aqueous periplasm. So far, efforts to mimic this environment under laboratory conditions have been limited by the complexity of the asymmetric bacterial outer membrane. To evade this impasse, we recently established a method to modify the protein composition of bacterial outer membrane vesicles (OMVs) released from Escherichia coli as a platform for biophysical studies of outer membrane proteins in their native membrane environment. Here, we apply protein-enriched OMVs to characterize the structure of three envelope proteins from E. coil using nuclear magnetic resonance (NMR) spectroscopy and expand the methodology to soluble periplasmic proteins. We obtain high-resolution in situ NMR spectra of the transmembrane protein OmpA as well as the periplasmic proteins CpxP and MalE. We find that our approach facilitates structural investigations of membrane-attached protein domains and is especially suited for soluble proteins within their native periplasmic environment. Thereby, the use of OMVs in solution NMR methods allows in situ analysis of the structure and dynamics of proteins twice the size compared to the current in-cell NMR methodology. We therefore expect our work to pave the way for more complex NMR studies of bacterial envelope proteins in the native environment of OMVs in the future.
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