α-Synuclein interaction with POPC/POPS vesicles.

We have investigated the adsorption of the amyloid-forming protein α-Synuclein (αSyn) onto small unilamellar vesicles composed of a mixture of zwitterionic POPC and anionic POPS lipids. αSyn monomers adsorb onto the anionic lipid vesicles where they adopt an α-helical secondary structure. The degree of adsorption depends on the fraction of anionic lipid in the mixed lipid membrane, but one needs to consider the electrostatic shift of the serine p with increasing fraction of POPS.

Structure and dynamics of the active site of hen egg-white lysozyme from atomic resolution neutron crystallography.

Hen egg-white lysozyme (HEWL) is a widely used model protein in crystallographic studies and its enzymatic mechanism has been extensively investigated for decades. Despite this, the interaction between the reaction intermediate and the catalytic Asp52, as well as the orientation of Asn44 and Asn46 side chains, remain ambiguous. Here, we report the crystal structures of perdeuterated HEWL and DO buffer-exchanged HEWL from 0.

Solution structure of the N-terminal extension domain of a asparaginyl-tRNA synthetase.

Several secreted proteins from helminths (parasitic worms) have been shown to have immunomodulatory activities. Asparaginyl-tRNA synthetases are abundantly secreted in the filarial nematode (AsnRS) and the parasitic flatworm (AsnRS), indicating a possible immune function. The suggestion is supported by AsnRS alleviating disease symptoms in a T-cell transfer mouse model of colitis.

Developing advanced models of biological membranes with hydrogenous and deuterated natural glycerophospholipid mixtures.

Cellular membranes are complex systems that consist of hundreds of different lipid species. Their investigation often relies on simple bilayer models including few synthetic lipid species. Glycerophospholipids (GPLs) extracted from cells are a valuable resource to produce advanced models of biological membranes.

High-Density Lipoprotein function is modulated by the SARS-CoV-2 spike protein in a lipid-type dependent manner.

There is a close relationship between the SARS-CoV-2 virus and lipoproteins, in particular high-density lipoprotein (HDL). The severity of the coronavirus disease 2019 (COVID-19) is inversely correlated with HDL plasma levels. It is known that the SARS-CoV-2 spike (S) protein binds the HDL particle, probably depleting it of lipids and altering HDL function.

Investigation on the relationship between lipid composition and structure in model membranes composed of extracted natural phospholipids.

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.

α-Synuclein Interaction with Lipid Bilayer Discs.

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.

Strikingly Different Roles of SARS-CoV-2 Fusion Peptides Uncovered by Neutron Scattering.

Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes.

Neutron crystallography reveals mechanisms used by Pseudomonas aeruginosa for host-cell binding.

The 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.

Cryo-EM structure of MsbA in saposin-lipid nanoparticles (Salipro) provides insights into nucleotide coordination.

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.

The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme.

The biological function of a protein is intimately related to its structure and dynamics, which in turn are determined by the way in which it has been folded. In vitro refolding is commonly used for the recovery of recombinant proteins that are expressed in the form of inclusion bodies and is of central interest in terms of the folding pathways that occur in vivo. Here, biophysical data are reported for in vitro-refolded hydrogenated hen egg-white lysozyme, in combination with atomic resolution X-ray diffraction analyses, which allowed detailed comparisons with native hydrogenated and refolded perdeuterated lysozyme.

On the Cluster Formation of α-Synuclein Fibrils.

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.

Elongation rate and average length of amyloid fibrils in solution using isotope-labelled small-angle neutron scattering.

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.

Structures of a deAMPylation complex rationalise the switch between antagonistic catalytic activities of FICD.

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.

SARS-CoV-2 spike protein removes lipids from model membranes and interferes with the capacity of high density lipoprotein to exchange lipids.

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.

ApoE and ApoE Nascent-Like HDL Particles at Model Cellular Membranes: Effect of Protein Isoform and Membrane Composition.

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.

Structural Characterization of Natural Yeast Phosphatidylcholine and Bacterial Phosphatidylglycerol Lipid Multilayers by Neutron Diffraction.

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.

Structural insights into protein folding, stability and activity using perdeuteration of hen egg-white lysozyme.

This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast , as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme.

Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis.

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.

Visualization of hydrogen atoms in a perdeuterated lectin-fucose complex reveals key details of protein-carbohydrate interactions.

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.

Apolipoprotein E Binding Drives Structural and Compositional Rearrangement of mRNA-Containing Lipid Nanoparticles.

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.

A Dimerization Site at SCR-17/18 in Factor H Clarifies a New Mechanism for Complement Regulatory Control.

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.

The Antifungal Mechanism of Amphotericin B Elucidated in Ergosterol and Cholesterol-Containing Membranes Using Neutron Reflectometry.

We have characterized and compared the structures of ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl--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.

Thermal Neutron Relative Biological Effectiveness Factors for Boron Neutron Capture Therapy from In Vitro Irradiations.

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.

Lipoprotein ability to exchange and remove lipids from model membranes as a function of fatty acid saturation and presence of cholesterol.

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.