129 results on '"Michael Haertlein"'
Search Results
2. Structures of a deAMPylation complex rationalise the switch between antagonistic catalytic activities of FICD
- Author
-
Luke A. Perera, Steffen Preissler, Nathan R. Zaccai, Sylvain Prévost, Juliette M. Devos, Michael Haertlein, and David Ron
- Subjects
Science - Abstract
The ER chaperone BiP is regulated by FICD-mediated AMPylation and deAMPylation. Here, the authors characterise the structure of mammalian AMPylated BiP bound to FICD, by X-ray crystallography and neutron scattering, providing insights into the mechanism of BiP AMPylation and deAMPylation.
- Published
- 2021
- Full Text
- View/download PDF
3. Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme
- Author
-
Joao Ramos, Valerie Laux, Michael Haertlein, Elisabetta Boeri Erba, Katherine E. McAuley, V. Trevor Forsyth, Estelle Mossou, Sine Larsen, and Annette E. Langkilde
- Subjects
perdeuterated lysozyme ,hewl ,isotope effect ,protein refolding ,biophysical characterization ,x-ray crystallography ,protein structure ,structural biology ,Crystallography ,QD901-999 - Abstract
This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris, as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97–Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.
- Published
- 2021
- Full Text
- View/download PDF
4. Nanoscale Structure and Dynamics of Model Membrane Lipid Raft Systems, Studied by Neutron Scattering Methods
- Author
-
Delaram Ahmadi, Katherine C. Thompson, Victoria García Sakai, Ralf Schweins, Martine Moulin, Michael Haertlein, Gernot A. Strohmeier, Harald Pichler, V. Trevor Forsyth, David J. Barlow, M. Jayne Lawrence, and Fabrizia Foglia
- Subjects
QENS ,SANS ,lipid rafts ,lipid ,multi-component systems ,Physics ,QC1-999 - Abstract
Quasi-elastic neutron scattering (QENS) and small angle neutron scattering (SANS), in combination with isotopic contrast variation, have been used to determine the structure and dynamics of three-component lipid membranes, in the form of vesicles, comprising an unsaturated [palmitoyl-oleoyl-phosphatidylcholine (POPC) or dioleoyl-phosphatidylcholine (DOPC)], a saturated phospholipid (dipalmitoyl-phosphatidylcholine (DPPC)), and cholesterol, as a function temperature and composition. SANS studies showed vesicle membranes composed of a 1:1:1 molar ratio of DPPC:DOPC:cholesterol and a 2:2:1 molar ratio of DPPC:POPC:cholesterol phase separated, forming lipid rafts of ∼18 and ∼7 nm diameter respectively, when decreasing temperature from 308 to 297 K. Phase separation was reversible upon increasing temperature. The larger rafts observed in systems containing DOPC are attributed to the greater mis-match in lipid alkyl chains between DOPC and DPPC, than for POPC and DPPC. QENS studies, over the temperature range 283–323K, showed that the resulting data were best modelled by two Lorentzian functions: a narrow component, describing the “in-plane” lipid diffusion, and a broader component, describing the lipid alkyl chain segmental relaxation. The overall “in-plane” diffusion was found to show a significant reduction upon increasing temperature due to the vesicle membranes transitioning from one containing rafts to one where the component lipids are homogeneously mixed. The use of different isotopic combinations allowed the measured overall reduction of in-plane diffusion to be understood in terms of an increase in diffusion of the saturated DPPC lipid and a corresponding decrease in diffusion of the unsaturated DOPC/POPC lipid. As the rafts are considered to be composed principally of saturated lipid and cholesterol, the breakdown of rafts decreases the exposure of the DPPC to cholesterol whilst increasing the exposure of cholesterol to unsaturated lipid. These results show the sensitivity of lipid diffusion to local cholesterol concentration, and the importance of considering the local, rather that the global composition of a membrane when understanding the diffusion processes of lipids within the membrane. The novel combination of SANS and QENS allows a non-intrusive approach to characterize the structure and dynamics occurring in phase-separated model membranes which are designed to mimic the lateral heterogeneity of lipids seen in cellular membranes–a heterogeneity that can have pathological consequences.
- Published
- 2022
- Full Text
- View/download PDF
5. On the Cluster Formation of α-Synuclein Fibrils
- Author
-
Marija Dubackic, Ilaria Idini, Veronica Lattanzi, Yun Liu, Anne Martel, Ann Terry, Michael Haertlein, Juliette M. Devos, Andrew Jackson, Emma Sparr, Sara Linse, and Ulf Olsson
- Subjects
alpha-synuclein ,amyloid fibril ,fractal cluster ,Lewy bodies (LB) ,small-angle neutron scattering (SANS) ,rigid-rod cluster modeling ,Biology (General) ,QH301-705.5 - Abstract
The dense accumulation of α-Synuclein fibrils in neurons is considered to be strongly associated with Parkinson’s disease. These intracellular inclusions, called Lewy bodies, also contain significant amounts of lipids. To better understand such accumulations, it should be important to study α-Synuclein fibril formation under conditions where the fibrils lump together, mimicking what is observed in Lewy bodies. In the present study, we have therefore investigated the overall structural arrangements of α-synuclein fibrils, formed under mildly acidic conditions, pH = 5.5, in pure buffer or in the presence of various model membrane systems, by means of small-angle neutron scattering (SANS). At this pH, α-synuclein fibrils are colloidally unstable and aggregate further into dense clusters. SANS intensities show a power law dependence on the scattering vector, q, indicating that the clusters can be described as mass fractal aggregates. The experimentally observed fractal dimension was d = 2.6 ± 0.3. We further show that this fractal dimension can be reproduced using a simple model of rigid-rod clusters. The effect of dominatingly attractive fibril-fibril interactions is discussed within the context of fibril clustering in Lewy body formation.
- Published
- 2021
- Full Text
- View/download PDF
6. A molecular mechanism for transthyretin amyloidogenesis
- Author
-
Ai Woon Yee, Matteo Aldeghi, Matthew P. Blakeley, Andreas Ostermann, Philippe J. Mas, Martine Moulin, Daniele de Sanctis, Matthew W. Bowler, Christoph Mueller-Dieckmann, Edward P. Mitchell, Michael Haertlein, Bert L. de Groot, Elisabetta Boeri Erba, and V. Trevor Forsyth
- Subjects
Science - Abstract
A number of disease-causing human transthyretin (TTR) mutations are known to lead to amyloid formation. Here the authors combine neutron crystallography, native mass spectrometry and modelling studies to characterize the T119M and S52P-TTR mutants, providing mechanistic insights into TTR amyloidosis.
- Published
- 2019
- Full Text
- View/download PDF
7. Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis
- Author
-
Khushboo Borah, Tom A Mendum, Nathaniel D Hawkins, Jane L Ward, Michael H Beale, Gerald Larrouy‐Maumus, Apoorva Bhatt, Martine Moulin, Michael Haertlein, Gernot Strohmeier, Harald Pichler, V Trevor Forsyth, Stephan Noack, Celia W Goulding, Johnjoe McFadden, and Dany J V Beste
- Subjects
chemostat ,metabolic flux ,metabolism ,Mycobacterium tuberculosis ,tuberculosis ,Biology (General) ,QH301-705.5 ,Medicine (General) ,R5-920 - Abstract
Abstract The co‐catabolism of multiple host‐derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady‐state chemostat system. We demonstrate that Mtb efficiently co‐metabolises either cholesterol or glycerol, in combination with two‐carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.
- Published
- 2021
- Full Text
- View/download PDF
8. ApoE and ApoE Nascent-Like HDL Particles at Model Cellular Membranes: Effect of Protein Isoform and Membrane Composition
- Author
-
Sarah Waldie, Federica Sebastiani, Martine Moulin, Rita Del Giudice, Nicolò Paracini, Felix Roosen-Runge, Yuri Gerelli, Sylvain Prevost, John C. Voss, Tamim A. Darwish, Nageshwar Yepuri, Harald Pichler, Selma Maric, V. Trevor Forsyth, Michael Haertlein, and Marité Cárdenas
- Subjects
ApoE isoforms ,lipid exchange ,reconstituted HDL ,model membranes ,neutron reflection ,small-angle neutron scattering ,Chemistry ,QD1-999 - Abstract
Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body.
- Published
- 2021
- Full Text
- View/download PDF
9. Structural Characterization of Natural Yeast Phosphatidylcholine and Bacterial Phosphatidylglycerol Lipid Multilayers by Neutron Diffraction
- Author
-
Alessandra Luchini, Giacomo Corucci, Krishna Chaithanya Batchu, Valerie Laux, Michael Haertlein, Viviana Cristiglio, and Giovanna Fragneto
- Subjects
natural lipids ,lipid multilayers ,neutron diffraction ,Pichia pastoris ,Escherichia coli ,deuterated lipids ,Chemistry ,QD1-999 - Abstract
Eukaryotic and prokaryotic cell membranes are difficult to characterize directly with biophysical methods. Membrane model systems, that include fewer molecular species, are therefore often used to reproduce their fundamental chemical and physical properties. In this context, natural lipid mixtures directly extracted from cells are a valuable resource to produce advanced models of biological membranes for biophysical investigations and for the development of drug testing platforms. In this study we focused on single phospholipid classes, i.e. Pichia pastoris phosphatidylcholine (PC) and Escherichia coli phosphatidylglycerol (PG) lipids. These lipids were characterized by a different distribution of their respective acyl chain lengths and number of unsaturations. We produced both hydrogenous and deuterated lipid mixtures. Neutron diffraction experiments at different relative humidities were performed to characterize multilayers from these lipids and investigate the impact of the acyl chain composition on the structural organization. The novelty of this work resides in the use of natural extracts with a single class head-group and a mixture of chain compositions coming from yeast or bacterial cells. The characterization of the PC and PG multilayers showed that, as a consequence of the heterogeneity of their acyl chain composition, different lamellar phases are formed.
- Published
- 2021
- Full Text
- View/download PDF
10. A Dimerization Site at SCR-17/18 in Factor H Clarifies a New Mechanism for Complement Regulatory Control
- Author
-
Orla M. Dunne, Xin Gao, Ruodan Nan, Jayesh Gor, Penelope J. Adamson, David L. Gordon, Martine Moulin, Michael Haertlein, V. Trevor Forsyth, and Stephen J. Perkins
- Subjects
analytical ultracentrifugation ,complement factor H ,inflammation ,molecular modelling ,X-ray scattering ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease.
- Published
- 2021
- Full Text
- View/download PDF
11. The Antifungal Mechanism of Amphotericin B Elucidated in Ergosterol and Cholesterol-Containing Membranes Using Neutron Reflectometry
- Author
-
Robin Delhom, Andrew Nelson, Valerie Laux, Michael Haertlein, Wolfgang Knecht, Giovanna Fragneto, and Hanna P. Wacklin-Knecht
- Subjects
amphotericin B ,lipid membranes ,POPC ,ergosterol ,cholesterol ,neutron reflection ,Chemistry ,QD1-999 - Abstract
We have characterized and compared the structures of ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes before and after interaction with the amphiphilic antifungal drug amphotericin B (AmB) using neutron reflection. AmB inserts into both pure POPC and sterol-containing membranes in the lipid chain region and does not significantly perturb the structure of pure POPC membranes. By selective per-deuteration of the lipids/sterols, we show that AmB extracts ergosterol but not cholesterol from the bilayers and inserts to a much higher degree in the cholesterol-containing membranes. Ergosterol extraction by AmB is accompanied by membrane thinning. Our results provide new insights into the mechanism and antifungal effect of AmB in these simple models of fungal and mammalian membranes and help understand the molecular origin of its selectivity and toxic side effects.
- Published
- 2020
- Full Text
- View/download PDF
12. Thermal Neutron Relative Biological Effectiveness Factors for Boron Neutron Capture Therapy from In Vitro Irradiations
- Author
-
María Pedrosa-Rivera, Javier Praena, Ignacio Porras, Manuel P. Sabariego, Ulli Köster, Michael Haertlein, V. Trevor Forsyth, José C. Ramírez, Clara Jover, Daniel Jimena, Juan L. Osorio, Patricia Álvarez, Carmen Ruiz-Ruiz, and María J. Ruiz-Magaña
- Subjects
boron neutron capture therapy ,relative biological effectiveness ,thermal neutrons ,Cytology ,QH573-671 - Abstract
The experimental determination of the relative biological effectiveness of thermal neutron factors is fundamental in Boron Neutron Capture Therapy. The present values have been obtained while using mixed beams that consist of both neutrons and photons of various energies. A common weighting factor has been used for both thermal and fast neutron doses, although such an approach has been questioned. At the nuclear reactor of the Institut Laue-Langevin a pure low-energy neutron beam has been used to determine thermal neutron relative biological effectiveness factors. Different cancer cell lines, which correspond to glioblastoma, melanoma, and head and neck squamous cell carcinoma, and non-tumor cell lines (lung fibroblast and embryonic kidney), have been irradiated while using an experimental arrangement designed to minimize neutron-induced secondary gamma radiation. Additionally, the cells were irradiated with photons at a medical linear accelerator, providing reference data for comparison with that from neutron irradiation. The survival and proliferation were studied after irradiation, yielding the Relative Biological Effectiveness that corresponds to the damage of thermal neutrons for the different tissue types.
- Published
- 2020
- Full Text
- View/download PDF
13. Formation and Characterization of Supported Lipid Bilayers Composed of Hydrogenated and Deuterated Escherichia coli Lipids.
- Author
-
Tania Kjellerup Lind, Hanna Wacklin, Jürgen Schiller, Martine Moulin, Michael Haertlein, Thomas Günther Pomorski, and Marité Cárdenas
- Subjects
Medicine ,Science - Abstract
Supported lipid bilayers are widely used for sensing and deciphering biomolecular interactions with model cell membranes. In this paper, we present a method to form supported lipid bilayers from total lipid extracts of Escherichia coli by vesicle fusion. We show the validity of this method for different types of extracts including those from deuterated biomass using a combination of complementary surface sensitive techniques; quartz crystal microbalance, neutron reflection and atomic force microscopy. We find that the head group composition of the deuterated and the hydrogenated lipid extracts is similar (approximately 75% phosphatidylethanolamine, 13% phosphatidylglycerol and 12% cardiolipin) and that both samples can be used to reconstitute high-coverage supported lipid bilayers with a total thickness of 41 ± 3 Å, common for fluid membranes. The formation of supported lipid bilayers composed of natural extracts of Escherichia coli allow for following biomolecular interactions, thus advancing the field towards bacterial-specific membrane biomimics.
- Published
- 2015
- Full Text
- View/download PDF
14. Mobility of a Mononucleotide within a Lipid Matrix: A Neutron Scattering Study
- Author
-
Loreto Misuraca, Francesca Natali, Laura da Silva, Judith Peters, Bruno Demé, Jacques Ollivier, Tilo Seydel, Valerie Laux-Lesourd, Michael Haertlein, Giuseppe Zaccai, David Deamer, and Marie Christine Maurel
- Subjects
neutron scattering ,multilamellar lipid matrix ,mononucleotide mobility ,hydration ,Science - Abstract
An essential question in studies on the origins of life is how nucleic acids were first synthesized and then incorporated into compartments about 4 billion years ago. A recent discovery is that guided polymerization within organizing matrices could promote a non-enzymatic condensation reaction allowing the formation of RNA-like polymers, followed by encapsulation in lipid membranes. Here, we used neutron scattering and deuterium labelling to investigate 5′-adenosine monophosphate (AMP) molecules captured in a multilamellar phospholipid matrix. The aim of the research was to determine and compare how mononucleotides are captured and differently organized within matrices and multilamellar phospholipid structures and to explore the role of water in organizing the system to determine at which level the system becomes sufficiently anhydrous to lock the AMP molecules into an organized structure and initiate ester bond synthesis. Elastic incoherent neutron scattering experiments were thus employed to investigate the changes of the dynamic properties of AMP induced by embedding the molecules within the lipid matrix. The influence of AMP addition to the lipid membrane organization was determined through diffraction measurement, which also helped us to define the best working Q range for dynamical data analysis with respect to specific hydration. The use of different complementary instruments allowed coverage of a wide time-scale domain, from ns to ps, of atomic mean square fluctuations, providing evidence of a well-defined dependence of the AMP dynamics on the hydration level.
- Published
- 2017
- Full Text
- View/download PDF
15. Production and analysis of perdeuterated lipids from Pichia pastoris cells.
- Author
-
Alexis de Ghellinck, Hubert Schaller, Valérie Laux, Michael Haertlein, Michele Sferrazza, Eric Maréchal, Hanna Wacklin, Juliette Jouhet, and Giovanna Fragneto
- Subjects
Medicine ,Science - Abstract
Probing molecules using perdeuteration (i.e deuteration in which all hydrogen atoms are replaced by deuterium) is extremely useful in a wide range of biophysical techniques. In the case of lipids, the synthesis of the biologically relevant unsaturated perdeuterated lipids is challenging and not usually pursued. In this work, perdeuterated phospholipids and sterols from the yeast Pichia pastoris grown in deuterated medium are extracted and analyzed as derivatives by gas chromatography and mass spectrometry respectively. When yeast cells are grown in a deuterated environment, the phospholipid homeostasis is maintained but the fatty acid unsaturation level is modified while the ergosterol synthesis is not affected by the deuterated culture medium. Our results confirm that the production of well defined natural unsaturated perdeuterated lipids is possible and gives also new insights about the process of desaturase enzymes.
- Published
- 2014
- Full Text
- View/download PDF
16. High-Density Lipoprotein function is modulated by the SARS-CoV-2 spike protein in a lipid-type dependent manner
- Author
-
Yubexi Correa, Rita Del Giudice, Sarah Waldie, Michel Thépaut, Samantha Micciula, Yuri Gerelli, Martine Moulin, Clara Delaunay, Franck Fieschi, Harald Pichler, Michael Haertlein, V. Trevor Forsyth, Anton Le Brun, Michael Moir, Robert A. Russell, Tamim Darwish, Jonas Brinck, Tigist Wodaje, Martin Jansen, César Martín, Felix Roosen - Runge, and Marité Cárdenas
- Subjects
Biomaterials ,Colloid and Surface Chemistry ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials - Published
- 2023
17. Developing advanced models of biological membranes with hydrogenous and deuterated natural glycerophospholipid mixtures
- Author
-
Giacomo Corucci, Krishna Chaithanya Batchu, Alessandra Luchini, Andreas Santamaria, Moritz Paul Karl Frewein, Valèrie Laux, Michael Haertlein, Yoshiki Yamaryo-Botté, Cyrille Y. Botté, Thomas Sheridan, Mark Tully, Armando Maestro, Anne Martel, Lionel Porcar, and Giovanna Fragneto
- Subjects
GC ,Monolayers ,Reflectometry ,SANS ,Glycerophospholipids ,SAXS ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Biomaterials ,HPTLC ,Colloid and Surface Chemistry ,Pichia pastoris ,Biomimetics ,Bilayers ,Deuteration ,Natural lipids ,HPLC - Published
- 2023
18. SARS-CoV-2 spike protein removes lipids from model membranes and interferes with the capacity of high density lipoprotein to exchange lipids
- Author
-
Yubexi Correa, V. Trevor Forsyth, Harald Pichler, Michael Haertlein, Franck Fieschi, Sarah Waldie, Marité Cárdenas, Michel Thépaut, Martine Moulin, Samantha Micciulla, Malmö Högskola = Malmö University, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Institut Laue-Langevin (ILL), Graz University of Technology [Graz] (TU Graz), Faculty of Health and Society, Malmö University, and ILL
- Subjects
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,MESH: Spike Glycoprotein, Coronavirus ,02 engineering and technology ,010402 general chemistry ,medicine.disease_cause ,MESH: Lipoproteins, HDL ,01 natural sciences ,Neutron reflection ,Biomaterials ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,High-density lipoprotein ,medicine ,Humans ,MESH: COVID-19 ,MESH: SARS-CoV-2 ,Infrared spectroscopy ,ComputingMethodologies_COMPUTERGRAPHICS ,Coronavirus ,MESH: Humans ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,SARS-CoV-2 ,Cholesterol ,COVID-19 ,Spike Protein ,Regular Article ,Läkemedelskemi ,021001 nanoscience & nanotechnology ,Lipids ,MESH: Lipids ,SARS-CoV-2 spike protein ,0104 chemical sciences ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Cell biology ,Membrane ,chemistry ,Spike Glycoprotein, Coronavirus ,lipids (amino acids, peptides, and proteins) ,Medicinal Chemistry ,Lipoproteins, HDL ,0210 nano-technology ,Function (biology) ,Lipoprotein - Abstract
Graphical abstract, Cholesterol has been shown to affect the extent of coronavirus binding and fusion to cellular membranes. The severity of Covid-19 infection is also known to be correlated with lipid disorders. Furthermore, the levels of both serum cholesterol and high-density lipoprotein (HDL) decrease with Covid-19 severity, with normal levels resuming once the infection has passed. Here we demonstrate that the SARS-CoV-2 spike (S) protein interferes with the function of lipoproteins, and that this is dependent on cholesterol. In particular, the ability of HDL to exchange lipids from model cellular membranes is altered when co-incubated with the spike protein. Additionally, the S protein removes lipids and cholesterol from model membranes. We propose that the S protein affects HDL function by removing lipids from it and remodelling its composition/structure.
- Published
- 2021
19. α-Synuclein Interaction with Lipid Bilayer Discs
- Author
-
Marija Dubackic, Yun Liu, Elizabeth G. Kelley, Crispin Hetherington, Michael Haertlein, Juliette M. Devos, Sara Linse, Emma Sparr, and Ulf Olsson
- Subjects
Neurons ,Amyloid ,Lipid Bilayers ,Electrochemistry ,alpha-Synuclein ,General Materials Science ,Surfaces and Interfaces ,Condensed Matter Physics ,Spectroscopy - Abstract
α-Synuclein (aSyn) is a 140 residue long protein present in presynaptic termini of nerve cells. The protein is associated with Parkinson's disease, in which case it has been found to self-assemble into long amyloid fibrils forming intracellular inclusions that are also rich in lipids. Furthermore, its synaptic function is proposed to involve interaction with lipid membranes, and hence, it is of interest to understand aSyn-lipid membrane interactions in detail. In this paper we report on the interaction of aSyn with model membranes in the form of lipid bilayer discs. Using a combination of cryogenic transmission electron microscopy and small-angle neutron scattering, we show that circular discs undergo a significant shape transition after the adsorption of aSyn. When aSyn self-assembles into fibrils, aSyn molecules desorb from the bilayer discs, allowing them to recover to their original shape. Interestingly, the desorption process has an all-or-none character, resulting in a binary coexistence of circular bilayer discs with no adsorbed aSyn and deformed bilayer discs having a maximum amount of adsorbed protein. The observed coexistence is consistent with the recent finding of cooperative aSyn adsorption to anionic lipid bilayers.
- Published
- 2022
20. Investigation on the relationship between lipid composition and structure in model membranes composed of extracted natural phospholipids
- Author
-
Andreas Santamaria, Krishna C. Batchu, Giovanna Fragneto, Valerie Laux, Michael Haertlein, Tamim A. Darwish, Robert A. Russell, Eduardo Guzman, Nathan Zaccai, Armando Maestro, Institut Laue-Langevin, Agence Nationale de la Recherche (France), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Ministerio de Ciencia e Innovación (España), Ikerbasque Basque Foundation for Science, Wellcome Trust, Eusko Jaurlaritza, and Australian Government
- Subjects
Biomaterials ,Neutron reflectometry ,Colloid and Surface Chemistry ,Model membranes ,Brewster angle microscopy ,Langmuir monolayers ,Extracted natural phospholipids ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials - Abstract
[Hypothesis] Unravelling the structural diversity of cellular membranes is a paramount challenge in life sciences. In particular, lipid composition affects the membrane collective behaviour, and its interactions with other biological molecules., [Experiments] Here, the relationship between membrane composition and resultant structural features was investigated by surface pressure-area isotherms, Brewster angle microscopy and neutron reflectometry on in vitro membrane models of the mammalian plasma and endoplasmic-reticulum-Golgi intermediate compartment membranes in the form of Langmuir monolayers. Natural extracted yeast lipids were used because, unlike synthetic lipids, the acyl chain saturation pattern of yeast and mammalian lipids are similar., [Findings] The structure of the model membranes, orthogonal to the plane of the membrane, as well as their lateral packing, were found to depend strongly on their specific composition, with cholesterol having a major influence on the in-plane morphology, yielding a coexistence of liquid-order and liquid-disorder phases., The authors thank the Institut Laue-Langevin (DOI: 10.5291/ILL-DATA.DIR-215) for financial support and allocation of beamtime, the ILL D-Lab for provision of the hydrogenous and deuterated yeast cells and the Partnership for Soft Condensed Matter (PSCM) for the lab support and provision of lipid extracts (L-Lab). Part of the lipid extraction activity was funded by the ANR/NSF-PIRE project REACT (Research and Education in Active Coatings Technologies for Human Health) and the Ligue for Advanced Neutron Sources (LENS). E.G. and A.M. received financial support from MICINN (grants PID2019-106557GB-C21 and PID2021-129054NA-I00, respectively), N.R.Z. from the Wellcome Trust (grant 207455/Z/17/Z). A. M. also acknowledges the financial support received from the IKUR Strategy under the collaboration agreement between Ikerbasque Foundation and Materials Physics Center on behalf of the Department of Education of the Basque Government. The National Deuteration Facility in Australia is partly funded by The National Collaborative Research Infrastructure Strategy (NCRIS), an Australian Government initiative.
- Published
- 2022
21. Elongation rate and average length of amyloid fibrils in solution using isotope-labelled small-angle neutron scattering
- Author
-
V. Trevor Forsyth, Michael Haertlein, Tuomas P. J. Knowles, Gudrun Lotze, Ann E. Terry, Patrick Flagmeier, James Doutch, David M. Dias, Adam M. Squires, Martine Moulin, Han Yin, Ben J. Eves, Annela M. Seddon, Eves, Ben J [0000-0003-0181-2847], Flagmeier, Patrick [0000-0002-1204-5340], Knowles, Tuomas PJ [0000-0002-0016-3008], Lotze, Gudrun [0000-0001-7995-2693], Squires, Adam M [0000-0003-1396-467X], and Apollo - University of Cambridge Repository
- Subjects
Materials science ,Nucleation ,Analytical chemistry ,02 engineering and technology ,Neutron scattering ,Neurodegenerative ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Biochemistry ,03 medical and health sciences ,QD ,Neutron ,3404 Medicinal and Biomolecular Chemistry ,Molecular Biology ,QC ,030304 developmental biology ,0303 health sciences ,Isotope ,34 Chemical Sciences ,Scattering ,FOS: Clinical medicine ,Neurosciences ,Alzheimer's Disease including Alzheimer's Disease Related Dementias (AD/ADRD) ,021001 nanoscience & nanotechnology ,Small-angle neutron scattering ,Chemistry ,Deuterium ,Chemistry (miscellaneous) ,Elongation ,0210 nano-technology - Abstract
Funder: Boehringer Ingelheim Fonds, Funder: University of Bath, We demonstrate a solution method that allows both elongation rate and average fibril length of assembling amyloid fibrils to be estimated. The approach involves acquisition of real-time neutron scattering data during the initial stages of seeded growth, using contrast matched buffer to make the seeds effectively invisible to neutrons. As deuterated monomers add on to the seeds, the labelled growing ends give rise to scattering patterns that we model as cylinders whose increase in length with time gives an elongation rate. In addition, the absolute intensity of the signal can be used to determine the number of growing ends per unit volume, which in turn provides an estimate of seed length. The number of ends did not change significantly during elongation, demonstrating that any spontaneous or secondary nucleation was not significant compared with growth on the ends of pre-existing fibrils, and in addition providing a method of internal validation for the technique. Our experiments on initial growth of alpha synuclein fibrils using 1.2 mg ml-1 seeds in 2.5 mg ml-1 deuterated monomer at room temperature gave an elongation rate of 6.3 ± 0.5 Å min-1, and an average seed length estimate of 4.2 ± 1.3 μm.
- Published
- 2021
22. Visualization of hydrogen atoms in a perdeuterated lectin-fucose complex reveals key details of protein-carbohydrate interactions
- Author
-
V. Trevor Forsyth, Anne Imberty, Matthew P. Blakeley, Michael Haertlein, Lukas Gajdos, Michaela Wimmerová, Juliette M. Devos, Atul Kumar, Centre de Recherches sur les Macromolécules Végétales (CERMAV), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Institut Laue-Langevin (ILL), ILL, Central European Institute of Technology [Brno] (CEITEC MU), and Brno University of Technology [Brno] (BUT)
- Subjects
Glycan ,Stereochemistry ,Stacking ,Q1 ,Fucose ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Structural Biology ,Lectins ,Aromatic amino acids ,Protein–carbohydrate interactions ,[CHIM]Chemical Sciences ,QD ,Binding site ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,Hydrogen bond ,Chemistry ,030302 biochemistry & molecular biology ,Ligand (biochemistry) ,3. Good health ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Photorhabdus ,Hydrogen ,Protein Binding ,QD415 - Abstract
Summary Carbohydrate-binding proteins from pathogenic bacteria and fungi have been shown to be implicated in various pathological processes, where they interact with glycans present on the surface of the host cells. These interactions are part of the initial processes of infection of the host and are very important to study at the atomic level. Here, we report the room temperature neutron structures of PLL lectin from Photorhabdus laumondii in its apo form and in complex with deuterated L-fucose, which is, to our knowledge, the first neutron structure of a carbohydrate-binding protein in complex with a fully deuterated carbohydrate ligand. A detailed structural analysis of the lectin-carbohydrate interactions provides information on the hydrogen bond network, the role of water molecules, and the extent of the CH-π stacking interactions between fucose and the aromatic amino acids in the binding site.
- Published
- 2021
23. Author response for 'Cryo‐EM structure of MsbA in saposin‐lipid nanoparticles (Salipro) provides insights into nucleotide coordination'
- Author
-
null Dominique‐Maurice Kehlenbeck, null Daouda A.K. Traore, null Inokentijs Josts, null Simon Sander, null Martine Moulin, null Michael Haertlein, null Sylvain Prevost, null Trevor V. Forsyth, and null Henning Tidow
- Published
- 2021
24. Cryo-EM structure of MsbA in saposin-lipid nanoparticles (Salipro) provides insights into nucleotide coordination
- Author
-
Dominique‐Maurice Kehlenbeck, Daouda A.K. Traore, Inokentijs Josts, Simon Sander, Martine Moulin, Michael Haertlein, Sylvain Prevost, V. Trevor Forsyth, and Henning Tidow
- Subjects
Cryoelectron Microscopy ,Cell Biology ,Biochemistry ,Saposins ,Adenosine Diphosphate ,Bacterial Proteins ,Liposomes ,Nanoparticles ,QD ,ddc:610 ,Vanadates ,Molecular Biology ,QD415 - Abstract
The FEBS journal 289(10), 2959 - 2970 (2022). doi:10.1111/febs.16327, The ATP-binding cassette transporter MsbA is a lipid flippase, translocating lipid A, glycolipids, and lipopolysaccharides from the inner to the outer leaflet of the inner membrane of Gram-negative bacteria. It has been used as a model system for time-resolved structural studies as several MsbA structures in different states and reconstitution systems (detergent/nanodiscs/peptidiscs) are available. However, due to the limited resolution of the available structures, detailed structural information on the bound nucleotides has remained elusive. Here, we have reconstituted MsbA in saposin A–lipoprotein nanoparticles (Salipro) and determined the structure of ADP-vanadate-bound MsbA by single-particle cryo-electron microscopy to 3.5 Å resolution. This procedure has resulted in significantly improved resolution and enabled us to model all side chains and visualise detailed ADP-vanadate interactions in the nucleotide-binding domains. The approach may be applicable to other dynamic membrane proteins., Published by Wiley-Blackwell, Oxford [u.a.]
- Published
- 2021
25. Neutron crystallography reveals novel mechanisms used by Pseudomonas aeruginosa for host-cell binding
- Author
-
Juliette M. Devos, Anne Imberty, Matthew P. Blakeley, Lukas Gajdos, V. Trevor Forsyth, and Michael Haertlein
- Subjects
biology ,Pseudomonas aeruginosa ,Chemistry ,Low-barrier hydrogen bond ,Lectin ,chemistry.chemical_element ,Calcium ,medicine.disease_cause ,Ligand (biochemistry) ,Fucose ,Crystallography ,chemistry.chemical_compound ,Deuterium ,medicine ,biology.protein ,Molecule - Abstract
SummaryThe opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections.
- Published
- 2021
26. Structures of a deAMPylation complex rationalise the switch between antagonistic catalytic activities of FICD
- Author
-
David Ron, Luke A. Perera, Nathan R. Zaccai, Juliette M. Devos, Michael Haertlein, Steffen Preissler, Sylvain Prévost, Perera, Luke A [0000-0002-0032-1176], Preissler, Steffen [0000-0001-7936-9836], Zaccai, Nathan R [0000-0002-1476-2044], Prévost, Sylvain [0000-0002-6008-1987], Devos, Juliette M [0000-0001-5989-6794], Ron, David [0000-0002-3014-5636], Apollo - University of Cambridge Repository, Perera, Luke A. [0000-0002-0032-1176], Zaccai, Nathan R. [0000-0002-1476-2044], and Devos, Juliette M. [0000-0001-5989-6794]
- Subjects
82/29 ,genetic structures ,Amino Acid Motifs ,General Physics and Astronomy ,631/45/173 ,0302 clinical medicine ,Adenosine Triphosphate ,Chaperones ,Endoplasmic Reticulum Chaperone BiP ,Heat-Shock Proteins ,631/337/458 ,chemistry.chemical_classification ,0303 health sciences ,Multidisciplinary ,biology ,article ,Nucleotidyltransferases ,3. Good health ,Tetratricopeptide ,Enzyme mechanisms ,631/45/470/1981 ,82/1 ,Dimerization ,Stereochemistry ,145 ,Science ,13/106 ,13/109 ,631/337/470/1463 ,General Biochemistry, Genetics and Molecular Biology ,Catalysis ,82/80 ,03 medical and health sciences ,Residue (chemistry) ,Humans ,Adenylylation ,82/83 ,X-ray crystallography ,030304 developmental biology ,82/16 ,Endoplasmic reticulum ,Membrane Proteins ,General Chemistry ,Solution structure ,Adenosine Monophosphate ,Enzyme ,chemistry ,631/535/1266 ,Chaperone (protein) ,biology.protein ,Biocatalysis ,Protein Processing, Post-Translational ,030217 neurology & neurosurgery ,Post-translational modifications - Abstract
The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP’s ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD’s antagonistic activities, despite a similar mode of engagement of its two substrates — unmodified and AMPylated BiP., The ER chaperone BiP is regulated by FICD-mediated AMPylation and deAMPylation. Here, the authors characterise the structure of mammalian AMPylated BiP bound to FICD, by X-ray crystallography and neutron scattering, providing insights into the mechanism of BiP AMPylation and deAMPylation.
- Published
- 2021
27. Deuterium effects on human serum albumin in solution
- Author
-
John W. White, David Heß, Nicholas J. Fraser, Trevor Forsyth, Michael Haertlein, and Valerie Laux
- Subjects
Chromatography ,Deuterium ,Chemistry ,QH ,medicine ,Electrical and Electronic Engineering ,Condensed Matter Physics ,Human serum albumin ,Electronic, Optical and Magnetic Materials ,medicine.drug - Abstract
'No abstract'
- Published
- 2018
28. Structures of a deAMPylation complex rationalise the switch between antagonistic catalytic activities of FICD (14/96/109)
- Author
-
David Ron, Juliette M. Devos, Nathan R. Zaccai, Luke A. Perera, Sylvain Prévost, Michael Haertlein, and Steffen Preissler
- Subjects
chemistry.chemical_classification ,genetic structures ,biology ,Chemistry ,Stereochemistry ,Endoplasmic reticulum ,Solution structure ,Catalysis ,Tetratricopeptide ,Residue (chemistry) ,Enzyme ,Chaperone (protein) ,biology.protein ,Adenylylation - Abstract
The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present long-sought crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP’s ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD’s antagonistic activities, despite a similar mode of engagement of its two substrates — unmodified and AMPylated BiP.
- Published
- 2021
29. Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme
- Author
-
Valerie Laux, V. Trevor Forsyth, Michael Haertlein, Elisabetta Boeri Erba, Katherine McAuley, Estelle Mossou, Sine Larsen, Joao Ramos, Annette Eva Langkilde, Institut Laue-Langevin (ILL), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), DIAMOND Light source, IT University of Copenhagen (ITU), Department of Drug Design and Pharmacology [Copenhagen] (ILF), Faculty of Health and Medical Sciences, University of Copenhagen = Københavns Universitet (UCPH)-University of Copenhagen = Københavns Universitet (UCPH), MS platform of the Grenoble Instruct-ERIC center (ISBG, UAR 3518 CNRS-CEA-UGA-EMBL), Diamond Light Source for beamtime at I03 and MAX IV for beamtime at BioMAX, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), European Project: GR/R99393/01, European Project: EP/C015452/1, ILL, IT University of Copenhagen, and University of Copenhagen = Københavns Universitet (KU)-University of Copenhagen = Københavns Universitet (KU)
- Subjects
biophysical characterization ,perdeuterated lysozyme ,protein refolding ,010402 general chemistry ,Q1 ,01 natural sciences ,Biochemistry ,Pichia pastoris ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,structural biology ,General Materials Science ,Thermal stability ,protein structure ,030304 developmental biology ,X-ray crystallography ,chemistry.chemical_classification ,0303 health sciences ,Crystallography ,biology ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Protein dynamics ,HEWL ,General Chemistry ,Condensed Matter Physics ,biology.organism_classification ,Research Papers ,R1 ,0104 chemical sciences ,Enzyme ,chemistry ,Structural biology ,QD901-999 ,Biophysics ,Protein folding ,Lysozyme ,QD415 ,isotope effect - Abstract
Perdeuteration and in vitro refolding of hen egg-white lysozyme impact protein thermal stability and activity. Deuteration appears to primarily affect enzymatic function through changes in protein dynamics, while refolding contributes to a small decrease in protein thermal stability., This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris, as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97–Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.
- Published
- 2021
30. Metabolic flux partitioning between the TCA cycle and glyoxylate shunt combined with a reversible methyl citrate cycle provide nutritional flexibility for Mycobacterium tuberculosis
- Author
-
Jane L. Ward, V.T. Forsyth, Johnjoe McFadden, Celia W. Goulding, D.J.V. Beste, Michael Haertlein, Khushboo Borah, Martine Moulin, Gernot Strohmeier, Stephan Noack, Michael H. Beale, Harald Pichler, Gerald Larrouy-Maumus, Tom A. Mendum, Apoorva Bhatt, and Nathaniel D. Hawkins
- Subjects
Citric acid cycle ,chemistry.chemical_classification ,Metabolic pathway ,Enzyme ,Biochemistry ,Chemistry ,Glyoxylate cycle ,Metabolic network ,Assimilation (biology) ,Metabolism ,Chemostat - Abstract
The utilisation of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection thereby identifying the Mtb specific metabolic pathways and enzymes required for carbon co-metabolism as potential drug targets. Metabolic flux represents the final integrative outcome of many different levels of cellular regulation that contribute to the flow of metabolites through the metabolic network. It is therefore critical that we have an in-depth understanding of the rewiring of metabolic fluxes in different conditions. Here, we employed 13C-metabolic flux analysis using stable isotope tracers (13C and 2H) and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly on physiologically relevant carbon sources in a steady state chemostat. We demonstrate that Mtb is able to efficiently co-metabolise combinations of either cholesterol or glycerol along with C2 generating carbon substrates. The uniform assimilation of the carbon sources by Mtb throughout the network indicated no compartmentalization of metabolism in these conditions however there were substrate specific differences in metabolic fluxes. This work identified that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle as the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide new insights into the metabolic architecture that affords adaptability of Mtb to divergent carbon substrates.ImportanceEach year more than 1 million people die of tuberculosis (TB). Many more are infected but successfully diagnosed and treated with antibiotics, however antibiotic-resistant TB isolates are becoming ever more prevalent and so novel therapies are urgently needed that can effectively kill the causative agent. Mtb specific metabolic pathways have been identified as an important drug target in TB. However the apparent metabolic plasticity of this pathogen presents a major obstacle to efficient targeting of Mtb specific vulnerabilities and therefore it is critical to define the metabolic fluxes that Mtb utilises in different conditions. Here, we used 13C-metabolic flux analysis to measure the metabolic fluxes that Mtb uses whilst growing on potential in vivo nutrients. Our analysis identified selective use of the metabolic network that included the TCA cycle, glyoxylate shunt and methyl citrate cycle. The metabolic flux phenotypes determined in this study improves our understanding about the co-metabolism of multiple carbon substrates by Mtb identifying a reversible methyl citrate cycle and the glyoxylate shunt as the critical metabolic nodes which underlie the nutritional flexibility of Mtb.
- Published
- 2021
31. Apolipoprotein E Binding Drives Structural and Compositional Rearrangement of mRNA-Containing Lipid Nanoparticles
- Author
-
Venkata R. Krishnamurthy, Marianna Yanez Arteta, Michael Lerche, Martine Moulin, Michael Haertlein, Christian Lang, Lennart Lindfors, V. Trevor Forsyth, Harald Pichler, Tamim A. Darwish, Robert A. Russell, Federica Sebastiani, Ryan A. Bragg, Lionel Porcar, Marité Cárdenas, Charles S. Elmore, and Sarah Waldie
- Subjects
Apolipoprotein E ,Biodistribution ,Small interfering RNA ,Endosome ,General Physics and Astronomy ,Protein Corona ,02 engineering and technology ,lipid nanoparticles ,Q1 ,010402 general chemistry ,Physical Chemistry ,01 natural sciences ,Article ,small-angle scattering ,chemistry.chemical_compound ,Apolipoproteins E ,protein corona ,mRNA delivery ,Distribution (pharmacology) ,Tissue Distribution ,QD ,General Materials Science ,RNA, Messenger ,RNA, Small Interfering ,Fysikalisk kemi ,Messenger RNA ,Chemistry ,Cholesterol ,General Engineering ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,ddc:540 ,Biophysics ,Nanoparticles ,lipids (amino acids, peptides, and proteins) ,0210 nano-technology ,ApoE - Abstract
Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP’s plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.
- Published
- 2021
32. Production of perdeuterated fucose from glyco-engineered bacteria
- Author
-
Lukas, Gajdos, V Trevor, Forsyth, Matthew P, Blakeley, Michael, Haertlein, Anne, Imberty, Eric, Samain, and Juliette M, Devos
- Subjects
deuteration ,fucose ,Polysaccharides ,AcademicSubjects/SCI01000 ,engineering ,neutron scattering ,Escherichia coli ,Glycan Synthesis - Abstract
l-Fucose and l-fucose-containing polysaccharides, glycoproteins or glycolipids play an important role in a variety of biological processes. l-Fucose-containing glycoconjugates have been implicated in many diseases including cancer and rheumatoid arthritis. Interest in fucose and its derivatives is growing in cancer research, glyco-immunology, and the study of host–pathogen interactions. l-Fucose can be extracted from bacterial and algal polysaccharides or produced (bio)synthetically. While deuterated glucose and galactose are available, and are of high interest for metabolic studies and biophysical studies, deuterated fucose is not easily available. Here, we describe the production of perdeuterated l-fucose, using glyco-engineered Escherichia coli in a bioreactor with the use of a deuterium oxide-based growth medium and a deuterated carbon source. The final yield was 0.2 g L−1 of deuterated sugar, which was fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We anticipate that the perdeuterated fucose produced in this way will have numerous applications in structural biology where techniques such as NMR, solution neutron scattering and neutron crystallography are widely used. In the case of neutron macromolecular crystallography, the availability of perdeuterated fucose can be exploited in identifying the details of its interaction with protein receptors and notably the hydrogen bonding network around the carbohydrate binding site.
- Published
- 2020
33. Thermal Neutron Relative Biological Effectiveness Factors for Boron Neutron Capture Therapy from In Vitro Irradiations
- Author
-
Ulli Köster, Daniel Jimena, María José Ruiz-Magaña, V. Trevor Forsyth, Carmen Ruiz-Ruiz, M.P. Sabariego, Michael Haertlein, Juan L. Osorio, José C. Ramírez, María Pedrosa-Rivera, Javier Praena, Ignacio Porras, Clara Jover, and Patricia Álvarez
- Subjects
life_sciences_other ,inorganic chemicals ,Materials science ,Astrophysics::High Energy Astrophysical Phenomena ,Quantitative Biology::Tissues and Organs ,Physics::Medical Physics ,Boron Neutron Capture Therapy ,Radiation ,Q1 ,Relative biological effectiveness ,Article ,030218 nuclear medicine & medical imaging ,law.invention ,Quantitative Biology::Cell Behavior ,03 medical and health sciences ,0302 clinical medicine ,law ,relative biological effectiveness ,Neoplasms ,Humans ,Neutron ,QD ,Irradiation ,Nuclear Experiment ,lcsh:QH301-705.5 ,thermal neutrons ,Neutrons ,integumentary system ,Radiochemistry ,technology, industry, and agriculture ,General Medicine ,Neutron radiation ,Nuclear reactor ,Neutron temperature ,Thermal neutrons ,Neutron capture ,lcsh:Biology (General) ,Gamma Rays ,boron neutron capture therapy ,030220 oncology & carcinogenesis ,biological sciences ,lipids (amino acids, peptides, and proteins) - Abstract
The experimental determination of the relative biological effectiveness of thermal neutron factors is fundamental in Boron Neutron Capture Therapy. The present values have been obtained while using mixed beams that consist of both neutrons and photons of various energies. A common weighting factor has been used for both thermal and fast neutron doses, although such an approach has been questioned. At the nuclear reactor of the Institut Laue-Langevin a pure low-energy neutron beam has been used to determine thermal neutron relative biological effectiveness factors. Different cancer cell lines, which correspond to glioblastoma, melanoma, and head and neck squamous cell carcinoma, and non-tumor cell lines (lung fibroblast and embryonic kidney), have been irradiated while using an experimental arrangement designed to minimize neutron-induced secondary gamma radiation. Additionally, the cells were irradiated with photons at a medical linear accelerator, providing reference data for comparison with that from neutron irradiation. The survival and proliferation were studied after irradiation, yielding the Relative Biological Effectiveness that corresponds to the damage of thermal neutrons for the different tissue types., Asociacion Espanola Contra el Cancer (AECC) PS16163811PORR, Spanish MINECO FIS2015-69941-C2-1-P, Junta de Andalucia P11-FQM-8229, Campus of International Excellence BioTic P-BS-64, University of Granada Chair Neutrons for Medicine: the Spanish Fundacion ACS, Asociacion Capitan Antonio, Fundacion ACS, La Kuadrilla de Iznalloz, Sonriendo Se Puede Ganar
- Published
- 2020
- Full Text
- View/download PDF
34. Radiobiology data of melanoma cells after low-energy neutron irradiation and boron compound administration
- Author
-
María Pedrosa-Rivera, M. José Ruiz-Magaña, Patricia Álvarez, Ignacio Porras, Javier Praena, Manuel P. Sabariego, Ulli Köster, Michael Haertlein, V. Trevor Forsyth, Torsten Soldner, José C. Ramírez, Clara Jover, Daniel Jimena, Juan L. Osorio, Ian Postuma, Carmen Ruiz-Ruiz
- Published
- 2020
- Full Text
- View/download PDF
35. Structural basis for activation of plasma-membrane Ca2+-ATPase by calmodulin
- Author
-
Julius Nitsche, Charlotte Uetrecht, V. Trevor Forsyth, Johannes Heidemann, Sebastian Busch, Inokentijs Josts, Dmitri I. Svergun, Michael Haertlein, Selma Maric, Haydyn D. T. Mertens, Martine Moulin, and Henning Tidow
- Subjects
0301 basic medicine ,Medicin och hälsovetenskap ,Calmodulin ,Calcium pump ,ATPase ,Medicine (miscellaneous) ,chemistry.chemical_element ,Calcium ,Q1 ,Medical and Health Sciences ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,ddc:570 ,QD ,lcsh:QH301-705.5 ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,QH ,3. Good health ,QR ,030104 developmental biology ,Enzyme ,chemistry ,Membrane protein ,lcsh:Biology (General) ,Cytoplasm ,Biophysics ,biology.protein ,General Agricultural and Biological Sciences ,Homeostasis ,QD415 - Abstract
Communications biology 1(1), 206 (2018). doi:10.1038/s42003-018-0203-7, Plasma-membrane Ca$\mathrm{^{2+}}$-ATPases expel Ca$\mathrm{^{2+}}$ from the cytoplasm and are key regulators of Ca$\mathrm{^{2+}}$ homeostasis in eukaryotes. They are autoinhibited under low Ca$\mathrm{^{2+}}$ concentrations. Calmodulin (CaM)-binding to a unique regulatory domain releases the autoinhibition and activates the pump. However, the structural basis for this activation, including the overall structure of this calcium pump and its complex with calmodulin, is unknown. We previously determined the high-resolution structure of calmodulin in complex with the regulatory domain of the plasma-membrane Ca$\mathrm{^{2+}}$-ATPase ACA8 and revealed a bimodular mechanism of calcium control in eukaryotes. Here we show that activation of ACA8 by CaM involves large conformational changes. Combining advanced modeling of neutron scattering data acquired from stealth nanodiscs and native mass spectrometry with detailed dissection of binding constants, we present a structural model for the full-length ACA8 Ca$\mathrm{^{2+}}$ pump in its calmodulin-activated state illustrating a displacement of the regulatory domain from the core enzyme., Published by Springer Nature, London
- Published
- 2018
36. Radiobiology data of melanoma cells after low-energy neutron irradiation and boron compound administration
- Author
-
María, Pedrosa-Rivera, M José, Ruiz-Magaña, Patricia, Álvarez, Ignacio, Porras, Javier, Praena, Manuel P, Sabariego, Ulli, Köster, Michael, Haertlein, V Trevor, Forsyth, Torsten, Soldner, José C, Ramírez, Clara, Jover, Daniel, Jimena, Juan L, Osorio, Ian, Postuma, and Carmen, Ruiz-Ruiz
- Subjects
Boron Compounds ,Neutrons ,Cell Line, Tumor ,Humans ,Boron Neutron Capture Therapy ,Melanoma - Abstract
The cold neutron beam at the PF1b line at the Institut Laue-Langevin (ILL), without fast neutrons and a low contribution of gamma rays, is a very suitable facility to measure cell damage following low-energy neutron irradiation. The biological damage associated with the thermal and the boron doses can be obtained in order to evaluate the relative biological effectiveness (RBE) for Boron Neutron Capture Therapy. Three different experiments were carried out on the A375 melanoma cell line: the first one in a hospital LINAC, to obtain the reference radiation data, and the other two at the ILL, in which the damage to cells with and without boron compounds added was measured.
- Published
- 2019
37. Hierarchical Nanotube Self‐Assembly of DNA Minor Groove‐Binding Ligand DB921 via Alkali Halide Triggering
- Author
-
Wai Li Ling, David W. Boykin, W. David Wilson, Melissa Gray, Mizar Oliva, Juliette M. Devos, Estelle Mossou, Ryo Mizuta, Theyencheri Narayanan, Patrice Rannou, Deeksha Munnur, Adam Round, Ralf Schweins, Edward P. Mitchell, V. Trevor Forsyth, Michael Haertlein, Abdelbasset A. Farahat, Stephen Neidle, Jessica Webster, European Synchrotron Radiation Facility (ESRF), European Molecular Biology Laboratory [Heidelberg] (EMBL), Keele Univ, Fac Nat Sci, Keele ST5 5BG, Staffs, England, Institut Laue-Langevin (ILL), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Synthèse, Structure et Propriétés de Matériaux Fonctionnels (STEP), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ILL Grenoble, F-38042 Grenoble, France, Swiss Federal Research Institute WSL-AR, ILL, Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Nanotube ,Materials science ,Polymers and Plastics ,Halide ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Materials Chemistry ,[CHIM]Chemical Sciences ,[PHYS.COND]Physics [physics]/Condensed Matter [cond-mat] ,ComputingMilieux_MISCELLANEOUS ,[PHYS]Physics [physics] ,Small-angle X-ray scattering ,Organic Chemistry ,[CHIM.MATE]Chemical Sciences/Material chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,Ligand (biochemistry) ,Alkali metal ,DNA Minor Groove Binding ,0104 chemical sciences ,Crystallography ,[CHIM.POLY]Chemical Sciences/Polymers ,Transmission electron microscopy ,Self-assembly ,0210 nano-technology - Abstract
International audience
- Published
- 2019
38. Functional dynamics of photosynthetic cells useful for biosensor development
- Author
-
Daniela Russo a, b, Maya Dimova Lambreva c, Gaetano Campi c, Alessio De Francesco a, Christiane Alba Simionesco d, Pierre Sebban e, Michael Haertlein f, Martine Moulin f, and Giuseppina Rea c
- Subjects
photosynthesis ,D1 mutants ,protein dynamics ,neutron scattering ,biosensors - Abstract
Photosynthesis is receiving renewed interest due to the possibility to integrate whole plant cells or their photosynthetic active sub-components into optoelectronic devices such as biosensors and organic semiconductors. Photosynthetic assemblies use light energy to power electron transfer and charge separation across a charge-impermeable lipid membrane. One of the main working photosynthetic units is the pigment/protein complex photosystem II (PSII) which hosts, among others, the D1/D2 reaction center (RC) proteins, where most of the photosynthetic redox active components are located. Photonic energy is captured by the antenna systems and sequentially transferred towards the RC at the primary acceptor plastoquinone QA, a single-electron carrier tightly bound to the D2 protein. Thereafter, electrons are transferred from QA to the secondary plastoquinone QB, bound to the D1 subunit, via a non-heme iron. QB is a two-electron carrier and after full reduction and protonation to QBH2, it is displaced by another molecule of the plastoquinone pool [1]. Some classes of environmental pollutants are competitive inhibitors of QB binding and can interrupt the photosynthetic electron transport chain. This activity can be easily monitored and it is the working principle underlying the functioning of PSII-based biosensors for the detection of toxic compounds [2-4]. The primary goal of our studies is to determine how structural, dynamics and functional proprieties of natural and mutated photosynthetic D1 RC proteins in green algae influence the sequential electron transfer reactions leading to efficient photochemical energy conversion. A better understanding of these factors will help to identify the parameters underlying an increased performance in terms of protein stability and functional reliability for biosensoristic purposes and to design molecular systems mimicking the high efficiency of solar energy conversion in natural photosynthesis. While the structure and the function of those systems are known, the protein dynamics and its relationship with the activity is still a focus of interest. The understanding and the ability to modulate the existing relations between structure-dynamics-functionality through selective genetic mutation, is extremely important for the biotechnological applications and fundamental research studies. Recently, we measured the flexibility and diffusive dynamics of Chlamydomonas cells and thylakoids carrying both native and single point mutated D1 proteins using neutron spectroscopy. In particular, we analyzed a set of mutants having improved or reduced affinity for specific classes of herbicides, and increased tolerance to ionizing radiation [5-7] (Figure 1A). By combining elastic and quasi-elastic neutron scattering data with chlorophyll fluorescence measurements, we revealed that single aminoacid replacements in the D1- plastoquinone binding niche impair electron transfer efficiency, and notably affect the temperature dependence of the overall protein dynamics, inferring increased flexibility to the host membranes, expanding to the entire cells [8]. Furthermore, to get a more detailed dynamical picture and to better understand the relation with the activity of the photosynthetic RC, we completed the single particle dynamics experiment with a collective dynamics study by neutron Brillouin spectroscopy [9]. Results indicated that single point mutations affect collective density fluctuations in hydrating water providing important insight on the transmission of information possibly correlated to biological functionality (Figure 2). [1] J. M. Erickson et al., The Plant Cell, 1, 1989, 361-371. [2] I. Husu et al., Sens. Actuators. B Chem. 185, 2013, 321-330. [3] D.J. Swainsbury et al., Biosens. Bioelectron. 58, 2014, 172-178. [4] M. Chatzipetrou, et al., Electrochem. commun. 64, 2016, 46-50. [5] G. Rea et al., PLoS ONE 6, 2011, e16216. [6] M.D. Lambreva et al., PLoS ONE 8, 2013, e61851 [7] M Turemis et al., Sens. Actuators. B Chem. 241, 2017, 993-1001. [8] D. Russo et al., Biophys J, in press. [9] D. Russo et al., J. Phys. Chem. Lett. 7, 2016, 2429-2433. This work was supported by COST Action TD1102 and the Lazio Regional project n. 85-2017-15256.
- Published
- 2019
39. In Vivo Water Dynamics in Shewanella oneidensis Bacteria at High Pressure
- Author
-
Fabrizia Foglia, Rachael Hazael, Filip Meersman, Martin C. Wilding, Victoria García Sakai, Sarah Rogers, Livia E. Bove, Michael Marek Koza, Martine Moulin, Michael Haertlein, V. Trevor Forsyth, Paul F. McMillan
- Published
- 2019
- Full Text
- View/download PDF
40. Neutron reflection study of the interaction of the eukaryotic pore-forming actinoporin equinatoxin II with lipid membranes reveals intermediate states in pore formation
- Author
-
Biserka Bakrač Bremec, Gregor Anderluh, Raymond S. Norton, Michael Haertlein, Trevor Forsyth, Nejc Rojko, M. Moulin, and Hanna Wacklin
- Subjects
0301 basic medicine ,Lysis ,Membrane lipids ,Lipid Bilayers ,Biophysics ,Q1 ,Biochemistry ,Phase Transition ,Protein Structure, Secondary ,Membrane Lipids ,03 medical and health sciences ,Cnidarian Venoms ,Mutant protein ,Animals ,Lipid bilayer phase behavior ,Lipid bilayer ,Neutrons ,030102 biochemistry & molecular biology ,Perforin ,Chemistry ,technology, industry, and agriculture ,Cell Biology ,Lipids ,Sphingomyelins ,Crystallography ,Cholesterol ,Sea Anemones ,030104 developmental biology ,Membrane ,Covalent bond ,lipids (amino acids, peptides, and proteins) ,Dimyristoylphosphatidylcholine ,Sphingomyelin ,Porosity - Abstract
Equinatoxin II (EqtII), a eukaryotic pore-forming toxin, lyses cell membranes through a mechanism involving the insertion of its N-terminal α-helix into the membrane. EqtII pore formation is dependent on sphingomyelin (SM), although cholesterol (Chol) and membrane microdomains have also been suggested to enhance its activity. We have investigated the mechanism of EqtII binding and insertion by using neutron reflection to determine the structures of EqtII–membrane assemblies in situ. EqtII has several different modes of binding to membranes depending on the lipid composition. In pure dimyristoyl-phosphatidylcholine (DMPC) membranes, EqtII interacts weakly and reversibly with the lipid head groups in an orientation approximately parallel to the membrane surface. The presence of sphingomyelin (SM) gives rise to a more upright orientation of EqtII, but Chol is required for insertion into the core of the membrane. Cooling the EqtII–lipid assembly below the lipid phase transition temperature leads to deep water penetration and a significant reduction in the extension of the protein outside the membrane, indicating that phase-separation plays a role in EqtII pore-formation. An inactive double-cysteine mutant of EqtII in which the α-helix is covalently tethered to the rest of the protein, interacts only reversibly with all the membranes. Releasing the α-helix in situ by reduction of the disulphide bridge, however, causes the mutant protein to penetrate in DMPC–SM–Chol membranes in a manner identical to that of the wild-type protein. Our results help clarify the early steps in pore formation by EqtII and highlight the valuable information on protein–membrane interactions available from neutron reflection measurements.
- Published
- 2016
41. Lipoprotein ability to exchange and remove lipids from model membranes as a function of fatty acid saturation and presence of cholesterol
- Author
-
Tamim A. Darwish, Gernot Strohmeier, Tania Kjellerup Lind, Martin Malmsten, V. Trevor Forsyth, Harald Pichler, Marité Cárdenas, Kathryn L. Browning, Sarah Waldie, Michael Haertlein, Martine Moulin, Armando Maestro, Federica Sebastiani, Maximilian W. A. Skoda, Selma Maric, Nageshwar R. Yepuri, and Eva Bengtsson
- Subjects
0301 basic medicine ,LATERAL DIFFUSION ,Cell ,030204 cardiovascular system & hematology ,Q1 ,medicine.disease_cause ,chemistry.chemical_compound ,APOLIPOPROTEIN B-100 ,HDL-CHOLESTEROL ,Lipid removal ,0302 clinical medicine ,Phospholipids ,chemistry.chemical_classification ,Fatty Acids ,Biochemistry and Molecular Biology ,DIMYRISTOYLPHOSPHATIDYLCHOLINE ,Lipids ,Plaque, Atherosclerotic ,A-I ,Lipoproteins, LDL ,Cholesterol ,Membrane ,medicine.anatomical_structure ,Biochemistry ,LOW-DENSITY LIPOPROTEINS ,NEUTRON-SCATTERING ,lipids (amino acids, peptides, and proteins) ,Lipoproteins, HDL ,Saturation (chemistry) ,Lipoproteins ,Phospholipid ,Neutron reflection ,LDL ,03 medical and health sciences ,medicine ,Humans ,Molecular Biology ,Triglycerides ,Cell Membrane ,Fatty acid ,Cell Biology ,BILAYER ,DEUTERATION ,Atherosclerosis ,Dietary Fats ,QR ,Saturated fats ,030104 developmental biology ,chemistry ,Biokemi och molekylärbiologi ,Oxidative stress ,Lipoprotein - Abstract
Lipoproteins play a central role in the development of atherosclerosis. High and low-density lipoproteins (HDL and LDL), known as ‘good’ and ‘bad’ cholesterol, respectively, remove and/or deposit lipids into the artery wall. Hence, insight into lipid exchange processes between lipoproteins and cell membranes is of particular importance in understanding the onset and development of cardiovascular disease. In order to elucidate the impact of phospholipid tail saturation and the presence of cholesterol in cell membranes on these processes, neutron reflection was employed in the present investigation to follow lipid exchange with both HDL and LDL against model membranes. Mirroring clinical risk factors for the development of atherosclerosis, lower exchange was observed in the presence of cholesterol, as well as when using an unsaturated phospholipid, compared to faster exchange when using a fully saturated phospholipid. These results highlight the importance of membrane composition on the interaction with lipoproteins, chiefly the saturation level of the lipids and presence of cholesterol, and provide novel insight into factors of importance for build-up and reversibility of atherosclerotic plaque. In addition, the correlation between the results and well-established clinical risk factors suggests that the approach taken can be employed also for understanding a broader set of risk factors including, e.g., effects of triglycerides and oxidative stress, as well as local effects of drugs on atherosclerotic plaque formation.
- Published
- 2020
42. Adsorption of Denaturated Lysozyme at the Air-Water Interface: Structure and Morphology
- Author
-
Olga Yu. Milyaeva, Imre Varga, Andrea Tummino, Richard A. Campbell, Valerie Laux, V. Trevor Forsyth, Shi-Yow Lin, Boris A. Noskov, Michael M. Krycki, and Michael Haertlein
- Subjects
Brewster's angle ,010304 chemical physics ,Chemistry ,02 engineering and technology ,Surfaces and Interfaces ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,01 natural sciences ,symbols.namesake ,chemistry.chemical_compound ,Adsorption ,Gibbs isotherm ,Chemical engineering ,0103 physical sciences ,Electrochemistry ,symbols ,General Materials Science ,Surface layer ,Steady state (chemistry) ,Neutron reflectometry ,Lysozyme ,0210 nano-technology ,Guanidine ,Spectroscopy - Abstract
The application of protein deuteration and high flux neutron reflectometry has allowed a comparison of the adsorption properties of lysozyme at the air-water interface from dilute solutions in the absence and presence of high concentrations of two strong denaturants: urea and guanidine hydrochloride (GuHCl). The surface excess and adsorption layer thickness were resolved and complemented by images of the mesoscopic lateral morphology from Brewster angle microscopy. It was revealed that the thickness of the adsorption layer in the absence of added denaturants is less than the short axial length of the lysozyme molecule, which indicates deformation of the globules at the interface. Two-dimensional elongated aggregates in the surface layer merge over time to form an extensive network at the approach to steady state. Addition of denaturants in the bulk results in an acceleration of adsorption and an increase of the adsorption layer thickness. These results are attributed to incomplete collapse of the globules in the bulk from the effects of the denaturants as a result of interactions between remote amino acid residues. Both effects may be connected to an increase of the effective total volume of macromolecules due to the changes of their tertiary structure, that is, the formation of molten globules under the influence of urea and the partial unfolding of globules under the influence of GuHCl. In the former case, the increase of globule hydrophobicity leads to cooperative aggregation in the surface layer during adsorption. Unlike in the case of solutions without denaturants, the surface aggregates are short and wormlike, their size does not change with time, and they do not merge to form an extensive network at the approach to steady state. To the best of our knowledge, these are the first observations of cooperative aggregation in lysozyme adsorption layers.
- Published
- 2018
43. Localization of Cholesterol within Supported Lipid Bilayers Made of a Natural Extract of Tailor-Deuterated Phosphatidylcholine
- Author
-
Harald Pichler, Sarah Waldie, Michael Haertlein, Martine Moulin, Tania Kjellerup Lind, Selma Maric, V. Trevor Forsyth, Kathryn L. Browning, Marité Cárdenas, Gernot Strohmeier, and Alessandra Luchini
- Subjects
0301 basic medicine ,Lipid Bilayers ,02 engineering and technology ,Neutron scattering ,03 medical and health sciences ,chemistry.chemical_compound ,Phosphatidylcholine ,Electrochemistry ,Molecule ,General Materials Science ,Lipid bilayer ,Spectroscopy ,Chemistry ,Cholesterol ,Bilayer ,Surfaces and Interfaces ,Deuterium ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,030104 developmental biology ,Membrane ,Phosphatidylcholines ,Biophysics ,lipids (amino acids, peptides, and proteins) ,0210 nano-technology - Abstract
Cholesterol is an essential component of mammalian membranes and is known to induce a series of physicochemical changes in the lipid bilayer. Such changes include the formation of liquid-ordered phases with an increased thickness and a configurational order as compared to liquid-disordered phases. For saturated lipid membranes, cholesterol molecules localize close to the lipid head group-tail interface. However, the presence of polyunsaturated lipids was recently shown to promote relocation of cholesterol toward the inner interface between the two bilayer leaflets. Here, neutron reflection is used to study the location of cholesterol (both non-deuterated and per-deuterated versions are used) within supported lipid bilayers composed of a natural mixture of phosphatidylcholine (PC). The lipids were produced in a genetically modified strain of Escherichia coli and grown under specific deuterated conditions to give an overall neutron scattering length density (which depends on the level of deuteration) of the lipids matching that of D
- Published
- 2018
44. Perdeuteration of cholesterol for neutron scattering applications using recombinant Pichia pastoris
- Author
-
Richard A. Campbell, Melanie Hirz, Michael Haertlein, Selma Maric, V. Trevor Forsyth, Katherine C. Thompson, Gernot Strohmeier, Harald Pichler, Martine Moulin, and Adrian R. Rennie
- Subjects
0301 basic medicine ,Membrane permeability ,Perdeuteration ,Neutron scattering ,bcs ,Biochemistry ,Mass Spectrometry ,Pichia ,Pichia pastoris ,03 medical and health sciences ,chemistry.chemical_compound ,Bioreactors ,Naturvetenskap ,Scattering, Small Angle ,QD ,Lipid engineering ,Lipid raft ,Molecular Biology ,biology ,Cholesterol ,Organic Chemistry ,Biochemistry and Molecular Biology ,Cell Biology ,biology.organism_classification ,Deuterium ,Recombinant Proteins ,Neutron Diffraction ,030104 developmental biology ,Membrane ,chemistry ,Biophysics ,lipids (amino acids, peptides, and proteins) ,Neutron reflectometry ,Natural Sciences ,Biokemi och molekylärbiologi - Abstract
Deuteration of biomolecules has a major impact on both quality and scope of neutron scattering experiments. Cholesterol is a major component of mammalian cells, where it plays a critical role in membrane permeability, rigidity and dynamics, and contributes to specific membrane structures such as lipid rafts. Cholesterol is the main cargo in low and high-density lipoprotein complexes (i.e. LDL, HDL) and is directly implicated in several pathogenic conditions such as coronary artery disease which leads to 17 million deaths annually. Neutron scattering studies on membranes or lipid-protein complexes exploiting contrast variation have been limited by the lack of availability of fully deuterated biomolecules and especially perdeuterated cholesterol. The availability of perdeuterated cholesterol provides a unique way of probing the structural and dynamical properties of the lipoprotein complexes that underly many of these disease conditions. Here we describe a procedure for in vivo production of perdeuterated recombinant cholesterol in lipid-engineered Pichia pastoris using flask and fed-batch fermenter cultures in deuterated minimal medium. Perdeuteration of the purified cholesterol was verified by mass spectrometry and its use in a neutron scattering study was demonstrated by neutron reflectometry measurements using the FIGAROO instrument at the ILL.
- Published
- 2018
45. Conformational States of ABC Transporter MsbA in a Lipid Environment Investigated by Small-Angle Scattering Using Stealth Carrier Nanodiscs
- Author
-
Haydyn D. T. Mertens, Michael Haertlein, Inokentijs Josts, Sylvain Prévost, V. Trevor Forsyth, Dmitri I. Svergun, Martine Moulin, Selma Maric, Sebastian Busch, Julius Nitsche, and Henning Tidow
- Subjects
0301 basic medicine ,Models, Molecular ,Materials science ,Neutron diffraction ,Genetic Vectors ,Lipid Bilayers ,Gene Expression ,ATP-binding cassette transporter ,Neutron scattering ,Protein Structure, Secondary ,03 medical and health sciences ,Bacterial Proteins ,X-Ray Diffraction ,Structural Biology ,Scattering, Small Angle ,Escherichia coli ,Cloning, Molecular ,Molecular Biology ,Integral membrane protein ,Nanodisc ,Small-angle X-ray scattering ,Scattering ,Membrane Proteins ,Deuterium ,Recombinant Proteins ,Nanostructures ,Neutron Diffraction ,030104 developmental biology ,Biophysics ,bacteria ,ATP-Binding Cassette Transporters ,Small-angle scattering - Abstract
Structural studies of integral membrane proteins (IMPs) are challenging, as many of them are inactive or insoluble in the absence of a lipid environment. Here, we describe an approach making use of fractionally deuterium labeled "stealth carrier" nanodiscs that are effectively invisible to low-resolution neutron diffraction and enable structural studies of IMPs in a lipidic native-like solution environment. We illustrate the potential of the method in a joint small-angle neutron scattering (SANS) and X-ray scattering (SAXS) study of the ATP-binding cassette (ABC) transporter protein MsbA solubilized in the stealth nanodiscs. The data allow for a direct observation of the signal from the solubilized protein without contribution from the surrounding lipid nanodisc. Not only the overall shape but also differences between conformational states of MsbA can be reliably detected from the scattering data, demonstrating the sensitivity of the approach and its general applicability to structural studies of IMPs.
- Published
- 2017
46. Lipid polyunsaturation determines the extent of membrane structural changes induced by Amphotericin B in Pichia pastoris yeast
- Author
-
Giovanna Fragneto, Valerie Laux, Michael Haertlein, Juliette Jouhet, Alexis de Ghellinck, Hanna Wacklin, Michele Sferrazza, Institut Laue-Langevin (ILL), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), European Spallation Source ESS AB, University of Copenhagen = Københavns Universitet (UCPH), European Project: 283883,EC:FP7:INFRA,FP7-INFRASTRUCTURES-2011-1,NMI3-II(2012), ILL, Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), University of Copenhagen = Københavns Universitet (KU), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Membrane Fluidity ,[SDV]Life Sciences [q-bio] ,Lipid Bilayers ,Biophysics ,Antifungal drug ,Phospholipid ,02 engineering and technology ,Biochemistry ,Pichia ,Pichia pastoris ,03 medical and health sciences ,chemistry.chemical_compound ,Amphotericin B ,Ergosterol ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Antifungal activity ,030304 developmental biology ,Yeast lipids ,Neutron reflectometry ,0303 health sciences ,biology ,Cell Membrane ,Polyunsaturation ,Fungi ,Membrane structure ,Cell Biology ,021001 nanoscience & nanotechnology ,biology.organism_classification ,Lipids ,Yeast ,Sterol ,Sterols ,Membrane interaction ,Membrane ,chemistry ,Membrane protein ,Fatty Acids, Unsaturated ,lipids (amino acids, peptides, and proteins) ,0210 nano-technology - Abstract
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbamem.2015.06.006.; International audience; The activity of the potent but highly toxic antifungal drug Amphotericin B (AmB), used intravenously to treat systemic fungal and parasitic infections, is widely accepted to result from its specific interaction with the fungal sterol ergosterol. While the effect of sterols on AmB activity has been intensely investigated, the role of membrane phospholipid composition has largely been ignored, and structural studies of native membranes have been hampered by their complex and disordered nature. We show for the first time that the structure of fungal membranes derived from Pichia pastoris yeast depends on the degree of lipid polyunsaturation, which has an impact on the structural consequences of AmB activity. AmB inserts in yeast membranes even in the absence of ergosterol, and forms an extra-membraneous layer whose thickness is resolved to be 4-5nm. In ergosterol-containing membranes, AmB insertion is accompanied by ergosterol extraction into this layer. The AmB-sponge mediated depletion of ergosterol from P. pastoris membranes gives rise to a significant membrane thinning effect that depends on the degree of lipid polyunsaturation. The resulting hydrophobic mismatch is likely to interfere with a much broader range of membrane protein functions than those directly involving ergosterol, and suggests that polyunsaturated lipids could boost the efficiency of AmB. Furthermore, a low degree of lipid polyunsaturation leads to least AmB insertion and may protect host cells against the toxic effects of AmB. These results provide a new framework based on lipid composition and membrane structure through which we can understand its antifungal action and develop better treatments.
- Published
- 2015
47. The aggregation of 'native' human serum albumin
- Author
-
David Heß, Valerie Laux, Anithahini Jeyasingham, John W. White, Trevor Forsyth, Jared K. Raynes, and Michael Haertlein
- Subjects
Heavy water ,Chromatography ,biology ,Biophysics ,Albumin ,General Medicine ,Fractionation ,Human serum albumin ,Phosphate ,Recombinant Proteins ,Protein Aggregates ,chemistry.chemical_compound ,chemistry ,biology.protein ,medicine ,Humans ,Bovine serum albumin ,Serum Albumin ,medicine.drug - Abstract
Recombinant fully deuterated, defatted human serum albumin in heavy water was found to be about 90 % aggregated before final fractionation. For comparison and to establish a datum for this isotope effect, the extent of aggregation is reported for “native” defatted and fatted human serum albumin solutions in phosphate buffered 1 mg/ml in heavy and light water at 25 °C and at 4 °C. The extent of aggregation is small over a month at these temperatures, but extensive when the solutions are subjected to repeated freeze-thawing from −18 to 25 °C in both D2O and H2O.
- Published
- 2015
48. Grafted biomembranes containing membrane proteins – the case of the leucine transporter
- Author
-
Marité Cárdenas, Kamil Gotfryd, Mikkel B. Thygesen, Vivien Jagalski, Selma Maric, Thomas Günther Pomorski, Michael Haertlein, Lei Shi, Claus J. Loland, Robert Barker, Nicolas Bovet, Mie Barthold Krüger, and Martine Moulin
- Subjects
Nitrilotriacetic Acid ,Amino Acid Transport Systems ,Sodium ,Detergents ,Lipid Bilayers ,chemistry.chemical_element ,Polyethylene glycol ,Molecular Dynamics Simulation ,Polyethylene Glycols ,chemistry.chemical_compound ,Phosphatidylcholine ,Naturvetenskap ,Amino acid transporter ,Lipid bilayer ,Chromatography ,Chemistry ,Nitrilotriacetic acid ,General Chemistry ,Condensed Matter Physics ,Membrane protein ,Phosphatidylcholines ,Quartz Crystal Microbalance Techniques ,Biophysics ,Gold ,Leucine ,Natural Sciences - Abstract
Here, we bind the sodium dependent amino acid transporter on nitrilotriacetic acid/polyethylene glycol functionalized gold sensors in detergents and perform a detergent–lipid exchange with phosphatidylcholine. We characterize the LeuT structure in the adsorbed film by magnetic contrast neutron reflection using the predicted model from molecular dynamic simulations.
- Published
- 2015
49. Neutrons describe ectoine effects on water H-bonding and hydration around a soluble protein and a cell membrane
- Author
-
Martine Moulin, Michael Haertlein, Anne L. Martel, Susanne von Gronau, François-Xavier Gallat, Dieter Oesterhelt, Gabriel J. Cuello, Bruno Demé, Irina Bagyan, Giuseppe Zaccai, Victor M. Galván Josa, Markus Neumann, Yann Fichou, Jérôme Combet, Martin Weik, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Bitop, Institut Charles Sadron (ICS), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Institut Laue-Langevin (ILL), Max-Planck-Institut für Biochemie = Max Planck Institute of Biochemistry (MPIB), Max-Planck-Gesellschaft, Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ILL, and Max-Planck-Institut für Biochemie (MPIB)
- Subjects
0301 basic medicine ,Neutron diffraction ,Ectoine ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Molecular dynamics ,Bacterial Proteins ,Scattering, Small Angle ,Escherichia coli ,ComputingMilieux_MISCELLANEOUS ,Multidisciplinary ,Aqueous solution ,030102 biochemistry & molecular biology ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Chemistry ,Cell Membrane ,Amino Acids, Diamino ,Water ,Hydrogen Bonding ,Deuterium ,Small-angle neutron scattering ,Neutron Diffraction ,030104 developmental biology ,Membrane ,Biochemistry ,13. Climate action ,Osmolyte ,Isotope Labeling ,Biophysics ,Halomonas - Abstract
Understanding adaptation to extreme environments remains a challenge of high biotechnological potential for fundamental molecular biology. The cytosol of many microorganisms, isolated from saline environments, reversibly accumulates molar concentrations of the osmolyte ectoine to counterbalance fluctuating external salt concentrations. Although they have been studied extensively by thermodynamic and spectroscopic methods, direct experimental structural data have, so far, been lacking on ectoine-water-protein interactions. In this paper, in vivo deuterium labeling, small angle neutron scattering, neutron membrane diffraction and inelastic scattering are combined with neutron liquids diffraction to characterize the extreme ectoine-containing solvent and its effects on purple membrane of H. salinarum and E. coli maltose binding protein. The data reveal that ectoine is excluded from the hydration layer at the membrane surface and does not affect membrane molecular dynamics, and prove a previous hypothesis that ectoine is excluded from a monolayer of dense hydration water around the soluble protein. Neutron liquids diffraction to atomic resolution shows how ectoine enhances the remarkable properties of H-bonds in water—properties that are essential for the proper organization, stabilization and dynamics of biological structures.
- Published
- 2017
50. Back-exchange of deuterium in neutron crystallography: characterization by IR spectroscopy
- Author
-
Matthew P. Blakeley, V. Trevor Forsyth, Michael Haertlein, Martine Moulin, Edward P. Mitchell, Ai Woon Yee, Keele Univ, Fac Nat Sci, Keele ST5 5BG, Staffs, England, Institut Laue-Langevin (ILL), ILL, and European Synchrotron Radiation Facility (ESRF)
- Subjects
0301 basic medicine ,deuteration ,Chemistry ,[SDV]Life Sciences [q-bio] ,Analytical chemistry ,Infrared spectroscopy ,Crystal growth ,Nuclear magnetic resonance crystallography ,Neutron scattering ,General Biochemistry, Genetics and Molecular Biology ,3. Good health ,Characterization (materials science) ,03 medical and health sciences ,Crystallography ,030104 developmental biology ,Deuterium ,neutron crystallography ,IR spectroscopy ,D2O/H2O back-exchange ,[CHIM]Chemical Sciences ,QD ,Neutron ,Laboratory Notes ,Neutron reflectometry - Abstract
The application of IR spectroscopy to the analysis of D2O/H2O back-exchange in samples used for neutron crystallography is described., The application of IR spectroscopy to the characterization and quality control of samples used in neutron crystallography is described. While neutron crystallography is a growing field, the limited availability of neutron beamtime means that there may be a delay between crystallogenesis and data collection. Since essentially all neutron crystallographic work is carried out using D2O-based solvent buffers, a particular concern for these experiments is the possibility of H2O back-exchange across reservoir or capillary sealants. This may limit the quality of neutron scattering length density maps and of the associated analysis. Given the expense of central facility beamtime and the effort that goes into the production of suitably sized (usually perdeuterated) crystals, a systematic method of exploiting IR spectroscopy for the analysis of back-exchange phenomena in the reservoirs used for crystal growth is valuable. Examples are given in which the characterization of D2O/H2O back-exchange in transthyretin crystals is described.
- Published
- 2017
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.