77 results on '"Bersch B"'
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2. Solution structure of the C-terminal domain of the vaccinia virus DNA polymerase processivity factor component A20 fused to a short peptide from the viral DNA polymerase E9.
- Author
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Bersch, B., primary, Tarbouriech, N., additional, Burmeister, W., additional, and Iseni, F., additional
- Published
- 2021
- Full Text
- View/download PDF
3. Solution structure of the C-terminal domain of the vaccinia virus DNA polymerase processivity factor component A20.
- Author
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Bersch, B., primary, Iseni, F., additional, Burmeister, W., additional, and Tarbouriech, N., additional
- Published
- 2021
- Full Text
- View/download PDF
4. Structural and metal binding characterization of SilB coordinations sites, a protein involved in metal resistance in Cupriavidus metallidurans CH34: SPE-5-4
- Author
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Vandenbussche, G., Fernández, P. U., and Bersch, B.
- Published
- 2014
5. Kontrastmittelechokardiographisch gesteuerte Perikarddrainage
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R. Erbel, Guido Caspari, Thomas Bartel, von Birgelen C, Krapp J, Bersch B, and S Möhlenkamp
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medicine.medical_specialty ,Percutaneous ,business.industry ,medicine.medical_treatment ,Pericardial cavity ,Pericardial fluid ,medicine.disease ,Hemopericardium ,Pericardial effusion ,Surgery ,Effusion ,Pericardiocentesis ,Cardiac tamponade ,medicine ,Radiology ,Cardiology and Cardiovascular Medicine ,business - Abstract
The most effective treatment for pericardial effusion and cardiac tamponade is removal of the pericardial fluid. Surgical pericardiotomy is associated with high mortality and morbidity. Similarly, subcostal percutaneous blind pericardiocentesis was reported to have unacceptably high mortality and complication rates. Major complications associated with blind needle punctures are right heart penetration, hemopericardium, puncture of the coronary arteries, liver and lung bleeding. Even under fluoroscopic guidance and electrocardiographic needle monitoring high complication rates persist. Pericardial drainage has been often inadequate, with frequent recurrences of significant pericardial effusions. Two-dimensional echocardiographically guided pericardiocentesis is reported to improve efficacy and safety of percutaneous puncture. Moreover, it allows immediate verification of the procedural success. We evaluated the efficacy and safety of an echocardiographically guided contrast agent controlled pericardiocentesis. This is a retrospective, descriptive study on 126 consecutive patients who underwent percutaneous pericardiocentesis at the University Hospital Essen, Germany, from 1995 to June 2000. There were 51 women (41%) and 75 men (55%) with a mean age of 52 +/- 14 years. Standard techniques for quantification of pericardial effusion were used. Depending on the localization of the pericardial effusion an apical or subxiphoidal approach was chosen. The puncture was performed under echocardiographic guidance and the position of the needle was controlled by injection of contrast agent. Over a long guidewire a pigtail catheter was inserted through a sheath for further drainage of pericardial fluid. The catheter was removed after a maximum of 48 hours to avoid infection of the pericardial cavity. An apical approach was chosen in 98 patients (78%), a subcostal in 28 patients (22%). The procedure was successful in 99% of the attempts. No death or clinical complication occurred. The maximal pericardial diameter measured by two-dimensional echocardiography was 32 +/- 16 mm before and 5.3 +/- 2 mm after drainage. The calculated pericardial effusion was 657 +/- 342 ml. A fluid volume of 605 +/- 342 ml could be drained. In all patients a pericardial catheter was placed for 1.4 +/- 0.8 days. Recurrence of pericardial effusion occurred in 18 patients (14%). Of these, 15 patients underwent repeated successful pericardiocentesis (2.5 +/- 0.8), and 3 patients were referred to surgical pericardiotomy. Pericardiocentesis under echocardiographic contrast agent guidance is a safe, successful and cost effective procedure for diagnostic and therapeutic drainage of pericardial effusion. Two-dimensional echocardiography allows localization of the optimal puncture site as well as the quantification of the effusion depth. The injection of contrast agents into the pericardial cavity improves the safety and accuracy of the procedure. Even recurrent pericardial effusions can be treated successfully.
- Published
- 2000
6. Bottom-up synthesis of vertically oriented two-dimensional materials
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Vilá, R A, primary, Momeni, K, additional, Wang, Q, additional, Bersch, B M, additional, Lu, N, additional, Kim, M J, additional, Chen, L Q, additional, and Robinson, J A, additional
- Published
- 2016
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7. NMR structure of a 180 residue construct encompassing the N-terminal metal-binding site and the membrane proximal domain of SilB from Cupriavidus metallidurans CH34
- Author
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Bersch, B., primary, Urbina Fernandez, P., additional, and Vandenbussche, G., additional
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- 2016
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8. N-terminal domain of pneumococcal PhtD protein with bound Zn(II)
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Bersch, B., primary, Bougault, C., additional, Favier, A., additional, Gabel, F., additional, Roux, L., additional, Vernet, T., additional, and Durmort, C., additional
- Published
- 2013
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9. The second dsRBD domain from A. thaliana DICER-LIKE 1
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Burdisso, P., primary, Suarez, I., additional, Bersch, B., additional, Bologna, N., additional, Palatnik, J., additional, Boisbouvier, J., additional, and Rasia, R., additional
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- 2013
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10. Solution structure of the C-terminal domain of SilB from Cupriavidus metallidurans
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Bersch, B., primary, Derfoufi, K., additional, and Vandenbussche, G., additional
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- 2011
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11. Cu(I)-bound CopK
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Bersch, B., primary
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- 2010
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12. Solution structure of CopK, a periplasmic protein involved in copper resistance in Cupriavidus metallidurans CH34
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Bersch, B., primary, Favier, A., additional, Schanda, P., additional, Coves, J., additional, van Aelst, S., additional, Vallaeys, T., additional, Wattiez, R., additional, and Mergeay, M., additional
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- 2008
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13. Characterization of substance P-membrane interaction by transferred nuclear Overhauser effect
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Augé, S., primary, Bersch, B., additional, Tropis, M., additional, and Milon, A., additional
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- 2000
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14. STRUCTURE OF THE EGF-LIKE MODULE OF HUMAN C1R, NMR, 19 STRUCTURES
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Bersch, B., primary, Hernandez, J.-F., additional, Marion, D., additional, and Arlaud, G.J., additional
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- 1997
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15. ECTOTHIORHODOSPIRA HALOPHILA CYTOCHROME C551 (REDUCED), NMR, 37 STRUCTURES
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Bersch, B., primary, Blackledge, M.J., additional, Meyer, T.E., additional, and Marion, D., additional
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- 1997
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16. Optimierung der Zerspanbarkeit von Automatenstahl.
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Bersch, B., Fröber, H., and Weise, H.
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- 1976
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17. Characterization of substance Pmembrane interaction by transferred nuclear Overhauser effect
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Augé, S., Bersch, B., Tropis, M., and Milon, A.
- Abstract
Substance P, one of the mammalian tachykinins, is known to interact strongly with lipid bilayers and this interaction may play a role in the receptorpeptide recognition process. The conformation of substance P bound to vesicles consisting of perdeuterated phosphatidylcholine has been investigated by means of two-dimensional transferred nuclear Overhauser (trNOE) spectroscopy. Nuclear magnetic resonance data analysis resulted in a unique conformational family characterized by a well-defined conformation of the last seven C-terminal amino acids, which consists of a sequence of nonstandard turns following each other in a helix-like manner. The absence of short- or medium-range trNOE in the N-terminal part indicates its structural flexibility. © 2000 John Wiley & Sons, Inc. Biopoly 54: 297306, 2000
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- 2000
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18. A conversation about HPV.
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Bersch B
- Published
- 2010
19. Oxonol VI as an optical indicator for membrane potentials in lipid vesicles
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Apell, H.-J, primary and Bersch, B, additional
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- 1987
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20. [Poxvirus-encoded DNA replication proteins: potential targets for antivirals].
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Tarbouriech N, Burmeister WP, Bersch B, and Iseni F
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- Humans, DNA, DNA Replication, Antiviral Agents pharmacology, Antiviral Agents therapeutic use, Poxviridae genetics, Variola virus genetics, Mpox (monkeypox)
- Abstract
In the spring of 2022, an epidemic due to human monkeypox virus (MPXV) of unprecedented magnitude spread across all continents. Although this event was surprising in its suddenness, the resurgence of a virus from the Poxviridae family is not surprising in a world population that has been largely naïve to these viruses since the eradication of the smallpox virus in 1980 and the concomitant cessation of vaccination. Since then, a vaccine and two antiviral compounds have been developed to combat a possible return of smallpox. However, the use of these treatments during the 2022 MPXV epidemic showed certain limitations, indicating the importance of continuing to develop the therapeutic arsenal against these viruses. For several decades, efforts to understand the molecular mechanisms involved in the synthesis of the DNA genome of these viruses have been ongoing. Although many questions remain unanswered up to now, the three-dimensional structures of essential proteins, and in particular of the DNA polymerase holoenzyme in complex with DNA, make it possible to consider the development of a model for poxvirus DNA replication. In addition, these structures are valuable tools for the development of new antivirals targeting viral genome synthesis. This review will first present the molecules approved for the treatment of poxvirus infections, followed by a review of our knowledge of the replication machinery of these viruses. Finally, we will describe how these proteins could be the target of new antiviral compounds.
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- 2024
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21. Assessment of prediction methods for protein structures determined by NMR in CASP14: Impact of AlphaFold2.
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Huang YJ, Zhang N, Bersch B, Fidelis K, Inouye M, Ishida Y, Kryshtafovych A, Kobayashi N, Kuroda Y, Liu G, LiWang A, Swapna GVT, Wu N, Yamazaki T, and Montelione GT
- Subjects
- Computational Biology, Machine Learning, Protein Folding, Sequence Analysis, Protein, Magnetic Resonance Spectroscopy methods, Membrane Proteins chemistry, Membrane Proteins metabolism, Models, Molecular, Protein Conformation, Software
- Abstract
NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis., (© 2021 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)
- Published
- 2021
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22. Scalable Characterization of 2D Gallium-Intercalated Epitaxial Graphene.
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El-Sherif H, Briggs N, Bersch B, Pan M, Hamidinejad M, Rajabpour S, Filleter T, Kim KW, Robinson J, and Bassim ND
- Abstract
Scalable synthesis of two-dimensional gallium (2D-Ga) covered by graphene layers was recently realized through confinement heteroepitaxy using silicon carbide substrates. However, the thickness, uniformity, and area coverage of the 2D-Ga heterostructures have not previously been studied with high-spatial resolution techniques. In this work, we resolve and measure the 2D-Ga heterostructure thicknesses using scanning electron microscopy (SEM). Utilizing multiple correlative methods, we find that SEM image contrast is directly related to the presence of uniform bilayer Ga at the interface and a variation of the number of graphene layers. We also investigate the origin of SEM contrast using both experimental measurements and theoretical calculations of the surface potentials. We find that a carbon buffer layer is detached due to the gallium intercalation, which increases the surface potential as an indication of the 2D-Ga presence. We then scale up the heterostructure characterization over a few-square millimeter area by segmenting SEM images, each acquired with nanometer-scale in-plane resolution. This work leverages the spectroscopic imaging capabilities of SEM that allows high-spatial resolution imaging for tracking intercalants, identifying relative surface potentials, determining the number of 2D layers, and further characterizing scalability and uniformity of low-dimensional materials.
- Published
- 2021
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23. How do Chaperones Bind (Partly) Unfolded Client Proteins?
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Sučec I, Bersch B, and Schanda P
- Abstract
Molecular chaperones are central to cellular protein homeostasis. Dynamic disorder is a key feature of the complexes of molecular chaperones and their client proteins, and it facilitates the client release towards a folded state or the handover to downstream components. The dynamic nature also implies that a given chaperone can interact with many different client proteins, based on physico-chemical sequence properties rather than on structural complementarity of their (folded) 3D structure. Yet, the balance between this promiscuity and some degree of client specificity is poorly understood. Here, we review recent atomic-level descriptions of chaperones with client proteins, including chaperones in complex with intrinsically disordered proteins, with membrane-protein precursors, or partially folded client proteins. We focus hereby on chaperone-client interactions that are independent of ATP. The picture emerging from these studies highlights the importance of dynamics in these complexes, whereby several interaction types, not only hydrophobic ones, contribute to the complex formation. We discuss these features of chaperone-client complexes and possible factors that may contribute to this balance of promiscuity and specificity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sučec, Bersch and Schanda.)
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- 2021
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24. Solution Structure of the C-terminal Domain of A20, the Missing Brick for the Characterization of the Interface between Vaccinia Virus DNA Polymerase and its Processivity Factor.
- Author
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Bersch B, Tarbouriech N, Burmeister WP, and Iseni F
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- Amino Acid Sequence, Catalytic Domain genetics, Crystallography, X-Ray, DNA, Viral chemistry, DNA, Viral genetics, DNA, Viral metabolism, DNA-Directed DNA Polymerase genetics, DNA-Directed DNA Polymerase metabolism, Holoenzymes chemistry, Holoenzymes genetics, Holoenzymes metabolism, Models, Molecular, Peptides chemistry, Peptides genetics, Peptides metabolism, Protein Binding, Sequence Homology, Amino Acid, Solutions chemistry, Vaccinia virus genetics, Viral Proteins genetics, Viral Proteins metabolism, Virus Replication genetics, DNA-Directed DNA Polymerase chemistry, Protein Domains, Vaccinia virus enzymology, Viral Proteins chemistry
- Abstract
Poxviruses are enveloped viruses with a linear, double-stranded DNA genome. Viral DNA synthesis is achieved by a functional DNA polymerase holoenzyme composed of three essential proteins. For vaccinia virus (VACV) these are E9, the catalytic subunit, a family B DNA polymerase, and the heterodimeric processivity factor formed by D4 and A20. The A20 protein links D4 to the catalytic subunit. High-resolution structures have been obtained for the VACV D4 protein in complex with an N-terminal fragment of A20 as well as for E9. In addition, biochemical studies provided evidence that a poxvirus-specific insertion (insert 3) in E9 interacts with the C-terminal residues of A20. Here, we provide solution structures of two different VACV A20 C-terminal constructs containing residues 304-426, fused at their C-terminus to either a BAP (Biotin Acceptor Peptide)-tag or a short peptide containing the helix of E9 insert 3. Together with results from titration studies, these structures shed light on the molecular interface between the catalytic subunit and the processivity factor component A20. The interface comprises hydrophobic residues conserved within the Chordopoxvirinae subfamily. Finally, we constructed a HADDOCK model of the VACV A20
304-426 -E9 complex, which is in excellent accordance with previous experimental data., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)- Published
- 2021
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25. Structural basis of client specificity in mitochondrial membrane-protein chaperones.
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Sučec I, Wang Y, Dakhlaoui O, Weinhäupl K, Jores T, Costa D, Hessel A, Brennich M, Rapaport D, Lindorff-Larsen K, Bersch B, and Schanda P
- Subjects
- Humans, Membrane Proteins metabolism, Mitochondrial Precursor Protein Import Complex Proteins, Scattering, Small Angle, X-Ray Diffraction, Mitochondrial Membranes metabolism, Molecular Chaperones chemistry
- Abstract
Chaperones are essential for assisting protein folding and for transferring poorly soluble proteins to their functional locations within cells. Hydrophobic interactions drive promiscuous chaperone-client binding, but our understanding of how additional interactions enable client specificity is sparse. Here, we decipher what determines binding of two chaperones (TIM8·13 and TIM9·10) to different integral membrane proteins, the all-transmembrane mitochondrial carrier Ggc1 and Tim23, which has an additional disordered hydrophilic domain. Combining NMR, SAXS, and molecular dynamics simulations, we determine the structures of Tim23/TIM8·13 and Tim23/TIM9·10 complexes. TIM8·13 uses transient salt bridges to interact with the hydrophilic part of its client, but its interactions to the transmembrane part are weaker than in TIM9·10. Consequently, TIM9·10 outcompetes TIM8·13 in binding hydrophobic clients, while TIM8·13 is tuned to few clients with both hydrophilic and hydrophobic parts. Our study exemplifies how chaperones fine-tune the balance of promiscuity versus specificity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)
- Published
- 2020
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26. Atomically thin half-van der Waals metals enabled by confinement heteroepitaxy.
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Briggs N, Bersch B, Wang Y, Jiang J, Koch RJ, Nayir N, Wang K, Kolmer M, Ko W, De La Fuente Duran A, Subramanian S, Dong C, Shallenberger J, Fu M, Zou Q, Chuang YW, Gai Z, Li AP, Bostwick A, Jozwiak C, Chang CZ, Rotenberg E, Zhu J, van Duin ACT, Crespi V, and Robinson JA
- Abstract
Atomically thin two-dimensional (2D) metals may be key ingredients in next-generation quantum and optoelectronic devices. However, 2D metals must be stabilized against environmental degradation and integrated into heterostructure devices at the wafer scale. The high-energy interface between silicon carbide and epitaxial graphene provides an intriguing framework for stabilizing a diverse range of 2D metals. Here we demonstrate large-area, environmentally stable, single-crystal 2D gallium, indium and tin that are stabilized at the interface of epitaxial graphene and silicon carbide. The 2D metals are covalently bonded to SiC below but present a non-bonded interface to the graphene overlayer; that is, they are 'half van der Waals' metals with strong internal gradients in bonding character. These non-centrosymmetric 2D metals offer compelling opportunities for superconducting devices, topological phenomena and advanced optoelectronic properties. For example, the reported 2D Ga is a superconductor that combines six strongly coupled Ga-derived electron pockets with a large nearly free-electron Fermi surface that closely approaches the Dirac points of the graphene overlayer.
- Published
- 2020
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27. The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments.
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Rampelt H, Sucec I, Bersch B, Horten P, Perschil I, Martinou JC, van der Laan M, Wiedemann N, Schanda P, and Pfanner N
- Subjects
- Biological Transport, Mitochondria metabolism, Mitochondrial Membrane Transport Proteins metabolism, Mitochondrial Membranes metabolism, Molecular Chaperones metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Background: The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway., Results: Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins., Conclusions: The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins.
- Published
- 2020
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28. Epitaxial graphene/silicon carbide intercalation: a minireview on graphene modulation and unique 2D materials.
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Briggs N, Gebeyehu ZM, Vera A, Zhao T, Wang K, De La Fuente Duran A, Bersch B, Bowen T, Knappenberger KL Jr, and Robinson JA
- Abstract
Intercalation of atomic species through epitaxial graphene on silicon carbide began only a few years following its initial report in 2004. The impact of intercalation on the electronic properties of the graphene is well known; however, the intercalant itself can also exhibit intriguing properties not found in nature. This realization has inspired new interest in epitaxial graphene/silicon carbide (EG/SiC) intercalation, where the scope of the technique extends beyond modulation of graphene properties to the creation of new 2D forms of 3D materials. The mission of this minireview is to provide a concise introduction to EG/SiC intercalation and to demonstrate a simplified approach to EG/SiC intercalation. We summarize the primary techniques used to achieve and characterize EG/SiC intercalation, and show that thermal evaporation-based methods can effectively substitute for more complex synthesis techniques, enabling large-scale intercalation of non-refractory metals and compounds including two-dimensional silver (2D-Ag) and gallium nitride (2D-GaN
x ).- Published
- 2019
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29. Structural Basis of Membrane Protein Chaperoning through the Mitochondrial Intermembrane Space.
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Weinhäupl K, Lindau C, Hessel A, Wang Y, Schütze C, Jores T, Melchionda L, Schönfisch B, Kalbacher H, Bersch B, Rapaport D, Brennich M, Lindorff-Larsen K, Wiedemann N, and Schanda P
- Subjects
- Amino Acid Sequence, Binding Sites, Intracellular Membranes metabolism, Mitochondrial Membrane Transport Proteins chemistry, Mitochondrial Membrane Transport Proteins genetics, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Protein Binding, Protein Domains, Protein Precursors chemistry, Protein Precursors metabolism, Protein Structure, Secondary, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins genetics, Sequence Alignment, Mitochondria metabolism, Mitochondrial Membrane Transport Proteins metabolism, Molecular Chaperones metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The exchange of metabolites between the mitochondrial matrix and the cytosol depends on β-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and β-barrel protein biogenesis. Our work reveals how a single mitochondrial "transfer-chaperone" system is able to guide α-helical and β-barrel membrane proteins in a "nascent chain-like" conformation through a ribosome-free compartment., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
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30. Dynamics and interactions of AAC3 in DPC are not functionally relevant.
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Kurauskas V, Hessel A, Dehez F, Chipot C, Bersch B, and Schanda P
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- Adenosine Diphosphate, Adenosine Triphosphate, Magnetic Resonance Spectroscopy, Membrane Transport Proteins, Mitochondrial ADP, ATP Translocases
- Published
- 2018
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31. How Detergent Impacts Membrane Proteins: Atomic-Level Views of Mitochondrial Carriers in Dodecylphosphocholine.
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Kurauskas V, Hessel A, Ma P, Lunetti P, Weinhäupl K, Imbert L, Brutscher B, King MS, Sounier R, Dolce V, Kunji ERS, Capobianco L, Chipot C, Dehez F, Bersch B, and Schanda P
- Subjects
- Mitochondrial ADP, ATP Translocases chemistry, Molecular Dynamics Simulation, Nuclear Magnetic Resonance, Biomolecular, Phosphorylcholine chemistry, Protein Conformation, Protein Stability, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae Proteins chemistry, Detergents chemistry, Micelles, Mitochondrial Membrane Transport Proteins chemistry, Phosphorylcholine analogs & derivatives
- Abstract
Characterizing the structure of membrane proteins (MPs) generally requires extraction from their native environment, most commonly with detergents. Yet, the physicochemical properties of detergent micelles and lipid bilayers differ markedly and could alter the structural organization of MPs, albeit without general rules. Dodecylphosphocholine (DPC) is the most widely used detergent for MP structure determination by NMR, but the physiological relevance of several prominent structures has been questioned, though indirectly, by other biophysical techniques, e.g., functional/thermostability assay (TSA) and molecular dynamics (MD) simulations. Here, we resolve unambiguously this controversy by probing the functional relevance of three different mitochondrial carriers (MCs) in DPC at the atomic level, using an exhaustive set of solution-NMR experiments, complemented by functional/TSA and MD data. Our results provide atomic-level insight into the structure, substrate interaction and dynamics of the detergent-membrane protein complexes and demonstrates cogently that, while high-resolution NMR signals can be obtained for MCs in DPC, they systematically correspond to nonfunctional states.
- Published
- 2018
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32. Functional reconstitution of cell-free synthesized purified K v channels.
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Renauld S, Cortes S, Bersch B, Henry X, De Waard M, and Schaack B
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- Elapid Venoms pharmacology, Escherichia coli genetics, Escherichia coli metabolism, Fluorescent Dyes chemistry, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Humans, Isoxazoles chemistry, Kv1.1 Potassium Channel genetics, Kv1.3 Potassium Channel genetics, Phosphatidylcholines chemistry, Phosphatidylcholines metabolism, Phosphatidylethanolamines chemistry, Phosphatidylethanolamines metabolism, Phosphatidylserines chemistry, Phosphatidylserines metabolism, Proteolipids chemistry, Proteolipids metabolism, Recombinant Proteins genetics, Recombinant Proteins metabolism, Subcellular Fractions metabolism, Valinomycin pharmacology, Kv1.1 Potassium Channel metabolism, Kv1.3 Potassium Channel metabolism, Membrane Potentials drug effects, Proteolipids drug effects
- Abstract
The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active K
v 1.1 and Kv 1.3 channels and their efficient insertion into liposomes. Two complementary methods for the determination of the electrical activity of the proteoliposome-embedded channels were compared using Kv 1.1 as a model system: (1) single channel recordings in droplet interface bilayers (DIB) and (2) measurement of the membrane voltage potential generated by a potassium ion diffusion potential using the voltage-sensitive fluorescent dye oxonol VI. Single channel recordings in DIBs proved unreliable because of the non-reproducible fusion of proteoliposomes with an artificial membrane. Therefore, the use of the optical indicator oxonol VI was adapted for 96 well microtiter plates using the ionophore valinomycin as a positive control. The activity of Kv 1.1 and Kv 1.3 channels was then monitored in the absence and presence of different venom toxins, demonstrating that fluorescent dyes can be used very efficiently when screening small molecules for their channel blocking activity., (Copyright © 2017 Elsevier B.V. All rights reserved.)- Published
- 2017
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33. Proton-Detected Solid-State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs.
- Author
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Bersch B, Dörr JM, Hessel A, Killian JA, and Schanda P
- Subjects
- Diffusion, Nanostructures chemistry, Proteolipids chemistry, Protons, Zinc chemistry, Bacterial Proteins chemistry, Cupriavidus chemistry, Maleates chemistry, Membrane Transport Proteins chemistry, Polystyrenes chemistry, Proton Magnetic Resonance Spectroscopy methods
- Abstract
The structure, dynamics, and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane and reconstituted in a suitable membrane-mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co-polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes to form nanosize discoidal proteolipid particles, also referred to as native nanodiscs. In this work, we show that high-resolution solid-state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified by the 2×34 kDa bacterial cation diffusion facilitator CzcD., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2017
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34. Structural and Functional Investigation of the Ag(+)/Cu(+) Binding Domains of the Periplasmic Adaptor Protein SilB from Cupriavidus metallidurans CH34.
- Author
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Urbina P, Bersch B, De Angelis F, Derfoufi KM, Prévost M, Goormaghtigh E, and Vandenbussche G
- Subjects
- Carrier Proteins metabolism, Cupriavidus metabolism, Ion Transport, Nuclear Magnetic Resonance, Biomolecular, Periplasm metabolism, Periplasmic Proteins metabolism, Protein Domains, Silver metabolism, Spectrometry, Fluorescence, Carrier Proteins chemistry, Cupriavidus chemistry, Periplasm chemistry, Periplasmic Proteins chemistry, Silver chemistry
- Abstract
Silver ion resistance in bacteria mainly relies on efflux systems, and notably on tripartite efflux complexes involving a transporter from the resistance-nodulation-cell division (RND) superfamily, such as the SilCBA system from Cupriavidus metallidurans CH34. The periplasmic adaptor protein SilB hosts two specific metal coordination sites, located in the N-terminal and C-terminal domains, respectively, that are believed to play a different role in the efflux mechanism and the trafficking of metal ions from the periplasm to the RND transporter. On the basis of the known domain structure of periplasmic adaptor proteins, we designed different protein constructs derived from SilB domains with either one or two metal binding sites per protein chain. ITC data acquired on proteins with single metal sites suggest a slightly higher affinity of Ag(+) for the N-terminal metal site, compared to that for the C-terminal one. Remarkably, via the study of a protein construct featuring both metal sites, nuclear magnetic resonance (NMR) and fluorescence spectroscopies concordantly show that the C-terminal site is saturated prior to the N-terminal one. The C-terminal binding site is supposed to transfer the metal ions to the RND protein, while the transport driven by this latter is activated upon binding of the metal ion to the N-terminal site. Our results suggest that the filling of the C-terminal metal site is a key prerequisite for preventing futile activation of the transport system. Exhaustive NMR studies reveal for the first time the structure and dynamics of the functionally important N-terminal domain connected to the membrane proximal domain as well as of its Ag(+) binding site.
- Published
- 2016
- Full Text
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35. Tungsten Ditelluride: a layered semimetal.
- Author
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Lee CH, Silva EC, Calderin L, Nguyen MA, Hollander MJ, Bersch B, Mallouk TE, and Robinson JA
- Abstract
Tungsten ditelluride (WTe2) is a transition metal dichalcogenide (TMD) with physical and electronic properties that make it attractive for a variety of electronic applications. Although WTe2 has been studied for decades, its structure and electronic properties have only recently been correctly described. We experimentally and theoretically investigate the structure, dynamics and electronic properties of WTe2, and verify that WTe2 has its minimum energy configuration in a distorted 1T structure (Td structure), which results in metallic-like transport. Our findings unambiguously confirm the metallic nature of WTe2, introduce new information about the Raman modes of Td-WTe2, and demonstrate that Td-WTe2 is readily oxidized via environmental exposure. Finally, these findings confirm that, in its thermodynamically favored Td form, the utilization of WTe2 in electronic device architectures such as field effect transistors may need to be reevaluated.
- Published
- 2015
- Full Text
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36. New insights into histidine triad proteins: solution structure of a Streptococcus pneumoniae PhtD domain and zinc transfer to AdcAII.
- Author
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Bersch B, Bougault C, Roux L, Favier A, Vernet T, and Durmort C
- Subjects
- Amino Acid Sequence, Apoproteins metabolism, Binding Sites, Biological Transport, Models, Molecular, Molecular Sequence Data, Protein Structure, Secondary, Solutions, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Carrier Proteins metabolism, Hydrolases chemistry, Hydrolases metabolism, Streptococcus pneumoniae metabolism, Zinc metabolism
- Abstract
Zinc (Zn(2+)) homeostasis is critical for pathogen host colonization and invasion. Polyhistidine triad (Pht) proteins, located at the surface of various streptococci, have been proposed to be involved in Zn(2+) homeostasis. The phtD gene, coding for a Zn(2+)-binding protein, is organized in an operon with adcAII coding for the extracellular part of a Zn(2+) transporter. In the present work, we investigate the relationship between PhtD and AdcAII using biochemical and structural biology approaches. Immuno-precipitation experiments on purified membranes of Streptococcus pneumoniae (S. pneumoniae) demonstrate that native PhtD and AdcAII interact in vivo confirming our previous in vitro observations. NMR was used to demonstrate Zn(2+) transfer from the Zn(2+)-bound form of a 137 amino acid N-terminal domain of PhtD (t-PhtD) to AdcAII. The high resolution NMR structure of t-PhtD shows that Zn(2+) is bound in a tetrahedral site by histidines 83, 86, and 88 as well as by glutamate 63. Comparison of the NMR parameters measured for apo- and Zn(2+)-t-PhtD shows that the loss of Zn(2+) leads to a diminished helical propensity at the C-terminus and increases the local dynamics and overall molecular volume. Structural comparison with the crystal structure of a 55-long fragment of PhtA suggests that Pht proteins are built from short repetitive units formed by three β-strands containing the conserved HxxHxH motif. Taken together, these results support a role for S. pneumoniae PhtD as a Zn(2+) scavenger for later release to the surface transporter AdcAII, leading to Zn(2+) uptake.
- Published
- 2013
- Full Text
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37. Second double-stranded RNA binding domain of dicer-like ribonuclease 1: structural and biochemical characterization.
- Author
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Burdisso P, Suarez IP, Bologna NG, Palatnik JF, Bersch B, and Rasia RM
- Subjects
- Arabidopsis enzymology, Arabidopsis Proteins metabolism, Cell Cycle Proteins metabolism, DEAD-box RNA Helicases chemistry, DNA metabolism, MicroRNAs metabolism, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Tertiary, Arabidopsis Proteins chemistry, Cell Cycle Proteins chemistry, RNA, Double-Stranded metabolism, Ribonuclease III chemistry, Ribonuclease III metabolism
- Abstract
Dicer-like ribonuclease III enzymes are involved in different paths related to RNA silencing in plants. Little is known about the structural aspects of these processes. Here we present a structural characterization of the second double-stranded RNA binding domain (dsRBD) of DCL1, which is presumed to participate in pri-micro-RNA recognition and subcellular localization of this protein. We determined the solution structure and found that it has a canonical fold but bears some variation with respect to other homologous domains. We also found that this domain binds both double-stranded RNA and double-stranded DNA, in contrast to most dsRBDs. Our characterization shows that this domain likely has functions other than substrate recognition and binding.
- Published
- 2012
- Full Text
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38. Structural and metal binding characterization of the C-terminal metallochaperone domain of membrane fusion protein SilB from Cupriavidus metallidurans CH34.
- Author
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Bersch B, Derfoufi KM, De Angelis F, Auquier V, Ekendé EN, Mergeay M, Ruysschaert JM, and Vandenbussche G
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Bacterial Proteins isolation & purification, Bacterial Proteins metabolism, Binding Sites, Copper metabolism, Membrane Fusion Proteins isolation & purification, Metallochaperones isolation & purification, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Peptide Fragments chemistry, Peptide Fragments metabolism, Protein Binding, Protein Structure, Tertiary, Sequence Alignment, Silver metabolism, Substrate Specificity, Cupriavidus, Membrane Fusion Proteins chemistry, Membrane Fusion Proteins metabolism, Metallochaperones chemistry, Metallochaperones metabolism, Metals metabolism
- Abstract
Detoxification of heavy metal ions in Proteobacteria is tightly controlled by various systems regulating their sequestration and transport. In Cupriavidus metallidurans CH34, a model organism for heavy metal resistance studies, the sil determinant is potentially involved in the efflux of silver and copper ions. Proteins SilA, SilB, and SilC form a resistance nodulation cell division (RND)-based transport system in which SilB is the periplasmic adaptor protein belonging to the membrane fusion protein (MFP) family. In addition to the four domains typical of known MFPs, SilB has a fifth additional C-terminal domain, called SilB(440-521), which is characterized here. Structure and backbone dynamics of SilB(440-521) have been investigated using nuclear magnetic resonance, and the residues of the metal site were identified from (15)N- and (13)C-edited HSQC spectra. The solution structure and additional metal binding experiments demonstrated that this C-terminal domain folds independently of the rest of the protein and has a conformation and a Ag(+) and Cu(+) binding specificity similar to those determined for CusF from Escherichia coli. The small protein CusF plays a role in metal trafficking in the periplasm. The similarity with CusF suggests a potential metallochaperone role for SilB(440-521) that is discussed in the context of simultaneous expression of different determinants involved in copper resistance in C. metallidurans CH34.
- Published
- 2011
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39. CopK from Cupriavidus metallidurans CH34 binds Cu(I) in a tetrathioether site: characterization by X-ray absorption and NMR spectroscopy.
- Author
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Sarret G, Favier A, Covès J, Hazemann JL, Mergeay M, and Bersch B
- Subjects
- Binding Sites, Ligands, Magnetic Resonance Spectroscopy, Models, Molecular, Protein Binding, Protein Conformation, Solutions, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Copper metabolism, Cupriavidus, Ether chemistry, X-Ray Absorption Spectroscopy
- Abstract
Cupriavidus metallidurans CH34 is a bacterium that is resistant to high metal concentrations in the environment. Increased copper resistance is associated with the cop cluster on the large plasmid pMOL30 that is composed of at least 21 genes. The copK gene encodes a 74 residue periplasmic protein whose expression is strongly upregulated in the presence of copper. CopK was previously shown to cooperatively bind Cu(I) and Cu(II) in distinct, specific sites. The solution structure of Cu(I)-CopK and the characterization of the Cu(I) site by X-ray absorption spectroscopy and NMR are reported here. EXAFS spectra are in agreement with a tetrathioether Cu(I) site, providing so far unique spectral information on a 4S-coordinated Cu(I) in a protein. The methionine residues forming the Cu(I) site, M28, M38, M44, and M54, are identified by NMR. We propose the chemical shift of the methionine C(epsilon) as a new and sensitive probe for the detection of Cu(I) bound to thioether groups. The solution structure of Cu(I)-CopK demonstrates that Cu(I) binding induces a complete structural modification with the disruption of the second beta-sheet and a rotation of the C-terminal part of nearly 180 degrees around a hinge formed by asparagine 57. This conformational change is directly related to the loss of the dimer interface and most probably to the formation of the Cu(II) site involving histidine 70. The solution structure of Cu(I)-CopK therefore provides the molecular basis for the understanding of the Cu(I)/Cu(II) binding cooperativity.
- Published
- 2010
- Full Text
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40. Site-directed mutagenesis reveals a conservation of the copper-binding site and the crucial role of His24 in CopH from Cupriavidus metallidurans CH34.
- Author
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Sendra V, Gambarelli S, Bersch B, and Covès J
- Subjects
- Binding Sites, Carrier Proteins genetics, Cupriavidus genetics, Histidine metabolism, Mutagenesis, Site-Directed, Mutation, Protein Binding, Carrier Proteins metabolism, Copper metabolism, Cupriavidus metabolism
- Abstract
CopH is a periplasmic copper-binding protein from Cupriavidus metallidurans CH34 that contains two histidine residues. Both His24 and His26 contribute to the formation of two high-affinity copper-binding sites in wild-type CopH and are likely involved in a 2N2O coordination sphere in the equatorial plane. We have used site-directed mutagenesis, and a series of spectroscopic and calorimetric studies to further characterize the copper-binding sites in CopH. While His24 plays a predominant role in copper affinity, one Cu-binding site was lost when either histidine residue was mutated. However, as shown by NMR and EPR, the mutation of the His residues does not affect the structural organization of the Cu-binding site nor the number of nitrogen ligands involved in copper ligation. In the absence of structural data, we propose a model that conciliates most of the spectroscopic data recorded during this study.
- Published
- 2009
- Full Text
- View/download PDF
41. Impact of selective excitation on carbon longitudinal relaxation: Towards fast solid-state NMR techniques.
- Author
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Giffard M, Bardet M, Bersch B, Covès J, and Hediger S
- Subjects
- Algorithms, Cupriavidus chemistry, DNA, Bacterial chemistry, Electron Spin Resonance Spectroscopy, Histidine chemistry, Isotope Labeling, Proteins chemistry, Carbon chemistry, Magnetic Resonance Spectroscopy methods
- Abstract
The effect of selective pulses on the apparent carbon longitudinal relaxation is investigated in three fully (13)C-labeled systems, histidine as a model system and two proteins MerP and YajG. It is shown that the longitudinal relaxation of a selectively excited carbon spin is greatly enhanced, mainly because of fast spin-diffusion. This relaxation enhancement allows reducing the time necessary for polarization recovery between two experiments. This effect can be exploited either to improve the sensitivity of NMR experiments or to reduce the experimental time. Using selective carbon excitation combined with fast pulsing on fully (13)C-labeled proteins, a sensitivity improvement of 20-45% over standard cross-polarization methods is predicted from the measured relaxation times.
- Published
- 2009
- Full Text
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42. Biostructural analysis of the metal-sensor domain of CnrX from Cupriavidus metallidurans CH34.
- Author
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Pompidor G, Girard E, Maillard A, Ramella-Pairin S, Bersch B, Kahn R, and Covès J
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Bacterial Proteins metabolism, Cloning, Molecular, Crystallization, Cupriavidus genetics, Dimerization, Gene Expression Regulation, Bacterial, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Periplasm metabolism, Sequence Analysis, DNA, Structure-Activity Relationship, X-Ray Diffraction, Bacterial Proteins chemistry, Cupriavidus drug effects, Cupriavidus metabolism, Drug Resistance, Bacterial, Metals, Heavy pharmacology, Signal Transduction
- Abstract
In Cupriavidus metallidurans CH34, the proteins CnrX, CnrY, and CnrH regulate the expression of the cnrCBA operon that codes for a cation-efflux pump involved in cobalt and nickel resistance. The periplasmic part of CnrX can be defined as the metal sensor in the signal transduction complex composed of the membrane-bound anti-sigma factor CnrY and the extra-cytoplasmic function sigma factor CnrH. A soluble form of CnrX was overproduced and purified. This protein behaves as a dimer in solution as judged from gel filtration, sedimentation velocity experiments, and NMR. Native crystals diffracting to 2.3 A using synchrotron radiation were obtained using the hanging-drop vapor-diffusion method. They belong to the primitive monoclinic space group P2(1), with unit cell parameters a = 31.87, b = 74.80, c = 93.67 A, beta = 90.107 degrees. NMR data and secondary structure prediction suggest that this protein is essentially formed by helices.
- Published
- 2009
- Full Text
- View/download PDF
43. Molecular structure and metal-binding properties of the periplasmic CopK protein expressed in Cupriavidus metallidurans CH34 during copper challenge.
- Author
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Bersch B, Favier A, Schanda P, van Aelst S, Vallaeys T, Covès J, Mergeay M, and Wattiez R
- Subjects
- Amino Acid Sequence, Animals, Bacterial Proteins genetics, Cupriavidus genetics, Gene Expression Regulation, Bacterial, Humans, Molecular Sequence Data, Molecular Structure, Nuclear Magnetic Resonance, Biomolecular, Periplasmic Proteins genetics, Protein Binding, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Copper metabolism, Cupriavidus chemistry, Periplasmic Proteins chemistry, Periplasmic Proteins metabolism, Protein Conformation
- Abstract
The copK gene is localized on the pMOL30 plasmid of Cupriavidus metallidurans CH34 within the complex cop cluster of genes, for which 21 genes have been identified. The expression of the corresponding periplasmic CopK protein is strongly upregulated in the presence of copper, leading to a high periplasmic accumulation. The structure and metal-binding properties of CopK were investigated by NMR and mass spectrometry. The protein is dimeric in the apo state with a dissociation constant in the range of 10(-5) M estimated from analytical ultracentrifugation. Mass spectrometry revealed that CopK has two high-affinity Cu(I)-binding sites per monomer with different Cu(I) affinities. Binding of Cu(II) was observed but appeared to be non-specific. The solution structure of apo-CopK revealed an all-beta fold formed of two beta-sheets in perpendicular orientation with an unstructured C-terminal tail. The dimer interface is formed by the surface of the C-terminal beta-sheet. Binding of the first Cu(I)-ion induces a major structural modification involving dissociation of the dimeric apo-protein. Backbone chemical shifts determined for the 1Cu(I)-bound form confirm the conservation of the N-terminal beta-sheet, while the last strand of the C-terminal sheet appears in slow conformational exchange. We hypothesize that the partial disruption of the C-terminal beta-sheet is related to dimer dissociation. NH-exchange data acquired on the apo-protein are consistent with a lower thermodynamic stability of the C-terminal sheet. CopK contains seven methionine residues, five of which appear highly conserved. Chemical shift data suggest implication of two or three methionines (Met54, Met38, Met28) in the first Cu(I) site. Addition of a second Cu(I) ion further increases protein plasticity. Comparison of the structural and metal-binding properties of CopK with other periplasmic copper-binding proteins reveals two conserved features within these functionally related proteins: the all-beta fold and the methionine-rich Cu(I)-binding site.
- Published
- 2008
- Full Text
- View/download PDF
44. Reversible redox- and zinc-dependent dimerization of the Escherichia coli fur protein.
- Author
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D'Autréaux B, Pecqueur L, Gonzalez de Peredo A, Diederix RE, Caux-Thang C, Tabet L, Bersch B, Forest E, and Michaud-Soret I
- Subjects
- Circular Dichroism, Cross-Linking Reagents, Dimerization, Disulfides, Oxidation-Reduction, Protein Conformation, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Bacterial Proteins chemistry, Escherichia coli Proteins chemistry, Repressor Proteins chemistry, Zinc chemistry
- Abstract
Fur is a bacterial regulator using iron as a cofactor to bind to specific DNA sequences. This protein exists in solution as several oligomeric states, of which the dimer is generally assumed to be the biologically relevant one. We describe the equilibria that exist between dimeric Escherichia coli Fur and higher oligomers. The dissociation constant for the dimer-tetramer equilibrium is estimated to be in the millimolar range. Oligomerization is enhanced at low ionic strength and pH. The as-isolated monomeric form of Fur is not in equilibrium with the dimer and contains two disulfide bridges (C92-C95 and C132-C137). Binding of the monomer to DNA is metal-dependent and sequence specific with an apparent affinity 5.5 times lower than that of the dimer. Size exclusion chromatography, EDC cross-linking, and CD spectroscopy show that reconstitution of the dimer from the monomer requires reduction of the disulfide bridges and coordination of Zn2+. Reduction of the disulfide bridges or Zn2+ alone does not promote dimerization. EDC and DMA cross-links reveal that the N-terminal NH2 group of one subunit is in an ionic interaction with acidic residues of the C-terminal tail and close to Lys76 and Lys97 of the other. Furthermore, the yields of cross-link drastically decrease upon binding of metal in the activation site, suggesting that the N-terminus is involved in the conformational change. Conversely, oxidizing reagents, H2O2 or diamide, disrupt the dimeric structure leading to monomer formation. These results establish that coordination of the zinc ion and the redox state of the cysteines are essential for holding E. coli Fur in a dimeric state.
- Published
- 2007
- Full Text
- View/download PDF
45. Structural changes of Escherichia coli ferric uptake regulator during metal-dependent dimerization and activation explored by NMR and X-ray crystallography.
- Author
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Pecqueur L, D'Autréaux B, Dupuy J, Nicolet Y, Jacquamet L, Brutscher B, Michaud-Soret I, and Bersch B
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray methods, DNA chemistry, Dimerization, Magnetic Resonance Spectroscopy methods, Models, Molecular, Molecular Sequence Data, Protein Denaturation, Protein Folding, Protein Structure, Quaternary, Protein Structure, Secondary, Sequence Homology, Amino Acid, Zinc chemistry, Bacterial Proteins chemistry, Escherichia coli metabolism, Repressor Proteins chemistry
- Abstract
Ferric uptake regulator (Fur) is a global bacterial regulator that uses iron as a cofactor to bind to specific DNA sequences. Escherichia coli Fur is usually isolated as a homodimer with two metal sites per subunit. Metal binding to the iron site induces protein activation; however the exact role of the structural zinc site is still unknown. Structural studies of three different forms of the Escherichia coli Fur protein (nonactivated dimer, monomer, and truncated Fur-(1-82)) were performed. Dimerization of the oxidized monomer was followed by NMR in the presence of a reductant (dithiothreitol) and Zn(II). Reduction of the disulfide bridges causes only local structure variations, whereas zinc addition to reduced Fur induces protein dimerization. This demonstrates for the first time the essential role of zinc in the stabilization of the quaternary structure. The secondary structures of the mono- and dimeric forms are almost conserved in the N-terminal DNA-binding domain, except for the first helix, which is not present in the nonactivated dimer. In contrast, the C-terminal dimerization domain is well structured in the dimer but appears flexible in the monomer. This is also confirmed by heteronuclear Overhauser effect data. The crystal structure at 1.8A resolution of a truncated protein (Fur-(1-82)) is described and found to be identical to the N-terminal domain in the monomeric and in the metal-activated state. Altogether, these data allow us to propose an activation mechanism for E. coli Fur involving the folding/unfolding of the N-terminal helix.
- Published
- 2006
- Full Text
- View/download PDF
46. CopH from Cupriavidus metallidurans CH34. A novel periplasmic copper-binding protein.
- Author
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Sendra V, Cannella D, Bersch B, Fieschi F, Ménage S, Lascoux D, and Covès J
- Subjects
- Amino Acid Sequence, Circular Dichroism, Cloning, Molecular, Dimerization, Electron Spin Resonance Spectroscopy, Mass Spectrometry, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Periplasm chemistry, Sequence Alignment, Spectrophotometry, Surface Plasmon Resonance, Burkholderiaceae chemistry, Carrier Proteins isolation & purification, Copper metabolism
- Abstract
The copH gene is one of the 19 open reading frames (ORFs) found in the cop cluster borne by the large plasmid pMol30 in Cupriavidus metallidurans CH34. The entire cluster is involved in detoxification of copper from the cytoplasm as well as from the periplasm. The function of the corresponding protein, CopH, is not yet clear, but it seems to be involved in the late response phase. We have cloned copH and overproduced and purified the corresponding protein. CopH is rather unique as only one paralog can be found in the databases. It is a dimeric protein with a molecular mass of 13 200 Da per subunit and located in the periplasm. The metal binding properties of CopH were examined by using a series of techniques such as UV-visible spectroscopy, circular dichroism (CD), electron paramagnetic resonance (EPR), surface plasmon resonance (SPR), mass spectrometry, and nuclear magnetic resonance (NMR). All together, the corresponding data are consistent with a dimeric protein containing one metal-binding site per subunit. These sites have a high affinity for Cu(II) but can also bind zinc or nickel. CopH does not contain any cysteines or methionines but contains two histidines. EPR and UV-visible features are consistent with the presence of Cu(II) type 2 centers in a nitrogen ligand field. SPR data confirm the involvement of the histidine residues in copper binding. CD and NMR data reveal that CopH is partially unfolded.
- Published
- 2006
- Full Text
- View/download PDF
47. Solution structure of the sulfite reductase flavodoxin-like domain from Escherichia coli.
- Author
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Sibille N, Blackledge M, Brutscher B, Covès J, and Bersch B
- Subjects
- Binding Sites, Crystallography, X-Ray, Electron Transport, Flavin Mononucleotide chemistry, Flavin Mononucleotide metabolism, Humans, Models, Molecular, Protein Folding, Protein Structure, Tertiary, Escherichia coli enzymology, Flavodoxin chemistry, Flavodoxin metabolism, Nuclear Magnetic Resonance, Biomolecular, Oxidoreductases Acting on Sulfur Group Donors chemistry, Oxidoreductases Acting on Sulfur Group Donors metabolism
- Abstract
The flavoprotein moiety of Escherichia coli sulfite reductase (SiR-FP) is homologous to electron transfer proteins such as cytochrome-P450 reductase (CPR) or nitric oxide synthase (NOS). We report on the three-dimensional structure of SiR-FP18, the flavodoxin-like domain of SiR-FP, which has been determined by NMR. In the holoenzyme, this domain plays an important role by shuttling electrons from the FAD to the hemoprotein (the beta-subunit). The structure presented here was determined using distance and torsion angle information in combination with residual dipolar couplings determined in two different alignment media. Several protein-FMN NOEs allowed us to place the prosthetic group in its binding pocket. The structure is well-resolved, and (15)N relaxation data indicate that SiR-FP18 is a compact domain. The binding interface with cytochrome c, a nonphysiological electron acceptor, has been determined using chemical shift mapping. Comparison of the SiR-FP18 structure with the corresponding domains from CPR and NOS shows that the fold of the protein core is highly conserved, but the analysis of the electrostatic surfaces reveals significant differences between the three domains. These observations are placed in the physiological context so they can contribute to the understanding of the electron transfer mechanism in the SiR holoenzyme.
- Published
- 2005
- Full Text
- View/download PDF
48. Biophysical characterization of the MerP-like amino-terminal extension of the mercuric reductase from Ralstonia metallidurans CH34.
- Author
-
Rossy E, Champier L, Bersch B, Brutscher B, Blackledge M, and Covès J
- Subjects
- Amino Acid Sequence, Circular Dichroism, Cloning, Molecular, Gene Expression, Mercury chemistry, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Oxidoreductases genetics, Oxidoreductases metabolism, Protein Binding, Protein Conformation, Proteins metabolism, Proteomics, Ralstonia genetics, Sequence Alignment, Spectrometry, Mass, Electrospray Ionization, Mercury metabolism, Oxidoreductases chemistry, Proteins chemistry, Ralstonia enzymology
- Abstract
The purified native mercuric reductase (MerA) from Ralstonia metallidurans CH34 contains an N-terminal sequence of 68 amino acids predicted to be homologous to MerP, the periplasmic mercury-binding protein. This MerP-like protein has now been expressed independently. The protein was named MerAa by homology with Ccc2a, the first soluble domain of the copper-transporting ATPase from yeast. Deltaa has been characterized using a set of biophysical techniques. The binding of mercury was followed using circular dichroism spectroscopy and electrospray mass spectrometry. The two cysteine residues contained in the consensus sequence GMTC XXC are involved in the binding of one mercury atom, with an apparent affinity comparable to that of MerP for the same metal. The metal-binding site is confirmed by NMR chemical shift changes observed between apo- and metal-bound MerAa in solution. NMR shift and NOE data also indicate that only minor structural changes occur upon metal binding. Further NMR investigation of the fold of MerAa using long-range methyl-methyl NOE and backbone residual dipolar coupling data confirm the expected close structural homology with MerP. (15)N relaxation data show that MerAa is a globally rigid molecule. An increased backbone mobility was observed for the loop region connecting the first beta-strand and the first alpha-helix and comprising the metal-binding domain. Although significantly reduced, this loop region keeps some conformational flexibility upon metal binding. Altogether, our data suggest a role of MerAa in mercury trafficking.
- Published
- 2004
- Full Text
- View/download PDF
49. Optimized set of two-dimensional experiments for fast sequential assignment, secondary structure determination, and backbone fold validation of 13C/15N-labelled proteins.
- Author
-
Bersch B, Rossy E, Covès J, and Brutscher B
- Subjects
- Models, Molecular, Nuclear Magnetic Resonance, Biomolecular, Protein Folding, Bacterial Proteins chemistry, Carbon chemistry, Nitrogen chemistry, Oxidoreductases chemistry, Protein Structure, Secondary
- Abstract
NMR experiments are presented which allow backbone resonance assignment, secondary structure identification, and in favorable cases also molecular fold topology determination from a series of two-dimensional 1H-15N HSQC-like spectra. The 1H-15N correlation peaks are frequency shifted by an amount +/- omegaX along the 15N dimension, where omegaX is the Calpha, Cbeta, or Halpha frequency of the same or the preceding residue. Because of the low dimensionality (2D) of the experiments, high-resolution spectra are obtained in a short overall experimental time. The whole series of seven experiments can be performed in typically less than one day. This approach significantly reduces experimental time when compared to the standard 3D-based methods. The here presented methodology is thus especially appealing in the context of high-throughput NMR studies of protein structure, dynamics or molecular interfaces.
- Published
- 2003
- Full Text
- View/download PDF
50. A recombinant chimeric epidermal growth factor-like module with high binding affinity for integrins.
- Author
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Vella F, Thielens NM, Bersch B, Arlaud GJ, and Frachet P
- Subjects
- Amino Acid Sequence, Animals, CHO Cells, Cell Adhesion, Cricetinae, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Protein Engineering, Recombinant Fusion Proteins chemistry, Surface Plasmon Resonance, Epidermal Growth Factor metabolism, Integrins metabolism, Recombinant Fusion Proteins metabolism
- Abstract
Integrins are cell surface receptors involved in numerous pathological processes such as metastasis invasion and abnormal angiogenesis. To target these receptors, the epidermal growth factor (EGF)-like domain of human complement protease C1r was used as a natural scaffold to design chimeric modules containing the RGD motif. Here we report a high yield bacterial expression system and its application to the production of two such modules, EGF-RGD and V2, the latter variant mimicking the RGD-containing domain of disintegrins. These modules were characterized chemically, and their biological activity was investigated by cellular assays using various Chinese hamster ovary cell lines expressing beta1 and beta3 integrins and by surface plasmon resonance spectroscopy. Remarkably, the modifications leading to the V2 variant had differential effects on the interaction with beta3 and beta1 integrins. The disintegrin-like V2 module exhibited enhanced binding affinities compared with EGF-RGD, with KD values of 7.2 nm for alpha5beta1 (a 4-fold decrease) and 3.5 nm for alphavbeta3 (a 1.5-fold decrease), comparable with the values determined for natural integrin ligands. Analysis by NMR spectroscopy also revealed a differential dynamic behavior of the RGD motif in the EGF-RGD and V2 variants, providing insights into the structural basis of their relative binding efficiency. These novel RGD-containing EGF modules open the way to the design of improved variants with selective affinity for particular integrins and their use as carriers for other biologically active modules.
- Published
- 2003
- Full Text
- View/download PDF
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