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Pseudomonas aeruginosa employs lectins to bind to its host cells, and is known to be the major cause of lung infections. Lectin B (LecB) from Pseudomonas aeruginosa binds specifically to galactose and fucose and is important for pathogenicity, adhesion and biofilm formation. In this work, the neutron crystal structure (1.9 Å) of the deuterated LecB/Ca/fucose complex is reported. The structure, in combination with perdeuteration of the ligand and the receptor allowed the observation of hydrogen atoms, protonation states and hydrogen bonds involved in the interaction between pathogenic bacteria and host cells. Thus the study provides structural insights into the mechanism of high affinity binding of LecB to its targets.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.