The NDF at the Australian Nuclear Science and Technology Organisation (ANSTO) provides deuteration through both biological and chemical techniques for a diversity of molecules and applications. Molecular deuteration of organic compounds and biomolecules significantly increases options in complex structure function investigations by providing contrast and improved data resolution when using...
Escherichia coli bacteria secrete colicins, a class of antibacterial proteins to kill closely related competing strains. This relies on the surprising ability of these toxins to cross the Gram-negative outer membrane (OM), a robust, impermeable, asymmetric lipid bilayer comprising an outer leaflet of lipopolysaccharide (LPS) and an inner leaflet of phospholipid. Colicins attach to their target...
Improving the parameters of biological macromolecules for usage in biomedicine and biotechnology requests the knowledge of the exact molecular mechanism of action of such biomolecules. Structural biology and dynamic studies of biological macromolecular complexes became essential part of such research. In addition to most abundant studies, where static structure of a protein is solved by the...
An important question in the study of chromatin packing is to identify the functional significance of its structure and dynamics. The integral properties of chromatin packing as a polymer chain are extremely important for understanding the mechanisms of interaction between distant chromatin regions, the formation of loops, and, ultimately, the formation and dynamics of topologically associated...
Most eukaryotic proteins are comprised of multiple structural domains connected by linkers of variable length and rigidity. We combine solution NMR spectroscopy and small angle scattering (SAXS, SANS) with crystallography and cryo-EM in integrative structural biology approaches to study the conformational dynamics of multidomain proteins and the roles of the connecting linkers. Studies with...
The SANS method is often used to study various types of biological systems. However, in the common case, SANS gives only a limited view on the structure of biomacromolecular complexes in solution. In this case, SANS can be used in conjunction with other methods, such as molecular modelling and molecular dynamics. Molecular modelling methods are also commonly used to study biomacromolecular...
IDPs are identified by the presence of unfolded region due to relatively
abundant polar residues content within its amino acid sequence. Together
with other residues, IDPs exhibit not only high flexibility but
also sensitivity to physico-chemical fluctuation such as pH, temperature,
and ions concentration. For this reason, IDPs are involved in
cellular processes such as DNA repair scheme...
Neutron techniques, incl. small angle neutron scattering (SANS), reflectometry (NR), protein crystallography (NPX), benefit from the usage of deuterated molecules. Many neutron facilities provide deuteration support to users to facilitate better measurements and enable research not possible without deuterated materials. NPX requires large protein crystals of partially or fully...
Neutrons techniques such as Neutron Small Angle Scattering (SANS), Neutron Macro-molecular Crystallography (NMX), Neutron Reflection (NR) are the popular non invasive probes in life sciences and health. All these neutronic instrumentation have been employed in studying the morphology of large molecules and macro-molecular complexes , the drug molecules and their interactions with the...
Dancing water molecules on the surface of soluble proteins provide the essential lubricant for macromolecular function. Surprisingly, polymers attached to protein surfaces have been reported to replace hydration water and bring inactive dry proteins back to life (Perriman et al. (2010) Nat Chem 2, 622; Gallat et al. (2012) JACS 134, 13168). The mechanism behind polymer-assisted protein motions...
Amyloid fibrils are self-assembled protein filaments, the deposition of which in tissues causes amyloidosis. Recently, much attention has been paid to polymorphism, where proteins form various amyloid fibrils that differ in structure and show different levels of cytotoxicity depending on fibrillation conditions. Since intramolecular motions in the fibrils are considered to play a crucial role...
Protein dynamics is pivotal to fulfill protein function. Apolipoprotein B-100 is a giant monomeric protein with a fascinating dynamical history: it mediates the conversion from very low density lipoprotein (VLDL, ~50 nm) to low density lipoprotein (LDL, 22 nm). As a key-player in the cholesterol transport system, the protein is intimately linked to the development of atherosclerosis and...
Intrinsically disordered proteins (IDPs) adopt a wide variety of conformations in solution, without a distinct equilibrium structure. Here, we investigate the dynamics of IDPs, using the antimicrobial saliva protein Histatin 5 as model. A suitable technique for this purpose is quasi- elastic neutron scattering (QENS), which through the incoherent scattering probes the self- diffusion of...
Hydrated proteins undergo a dynamical transition (DT) at $T_d$ ≈ 180-230 K. The transition is associated with the activation of protein dynamics on the ps-ns time scale, suitably detected by Elastic Incoherent Neutron Scattering (EINS). The DT has been also observed in other biomolecules and is deemed necessary for biological functionality. Surprisingly, a DT has been recently found in a...
Cancer is one of the major threats to our health on a global scale. In order to battle these diseases while maintaining the quality of life for patients it is important to find anticancer drugs with a high selectivity for the target cancer cell. Melittin, a peptide found in Honey bee venom has long been known for its antimicrobial effects. Later studies have also shown Melittin to be effective...
Clathrin-mediated endocytosis is a crucial cell biology process allowing internalization of many cell-surface proteins, and other cargo, in eukaryotes. Clathrin-coated vesicles (CCVs) are assembled with their cargo at the plasma membrane, then transport to the early endosome inside the cell. A CCV consists of a clathrin scaffold coating a lipid vesicle, in which the cargo is embedded, linked...
The neutron backscattering spectrometer SPHERES (SPectrometer for High Energy RESolution) at MLZ is a third generation backscattering spectrometer with focusing optics and phase-space transform (PST) chopper. It covers a dynamic range of ± 31μeV with a high resolution of about 0.66μeV and a good signal-to-noise ratio. The instrument performance has been improved over the recent years by...
Following molecular dynamics during the temporary evolution of kinetically changing samples is a major challenge. With recent developments of analysis frameworks, accessing the short-time self-diffusive properties of protein solutions by measuring specific energy transfers via neutron backscattering, kinetically changing samples can be investigated. The immobile fraction, determined by...
Extracellular vesicles (EVs) are a potent intercellular communication system. Such small vesicles transport biomolecules between cells and throughout the body, strongly influencing the fate of recipient cells. Due to their specific biological functions they have been proposed as biomarkers for various diseases and as optimal candidates for therapeutic applications. Despite of their extreme...
NADH-cytochrome b5 reductase (b5R) on endoplasmic reticulum membrane in mammalian liver cell plays a variety of roles concerning lipid unsaturation and xenobiotic metabolism. b5R transfers electrons from two-electron carrier of NADH to one-electron donor of cytochrome b5. In the redox cycle of b5R, a hydride transfer from NADH to oxidized FAD and deprotonation from the reduced FADH take place...
Casein kinase II (CK2), is a serine / threonine kinase ubiquitously distributed among eukaryotic cells, is known to be involved in the cell cycle and cell survival and proliferation. CK2 is one of the drug target proteins, because the relationship between CK2 over-expression and carcinogenesis and cancer metastasis has been pointed out. We aimed to elucidate the hydrogen bonding network...
Understanding the structure and dynamics of phospholipid membranes is of paramount importance for biophysics, biology and medical sciences. Virtually every living organisms is comprised of several of those membranes that provide a variety of functions, ranging from the separation of volumes to more complex functions like nutrient or information transport across the membrane.
All those...
Kai-clock system is one of the simplest biological clocks: the system is composed with only three proteins, KaiA, KaiB and KaiC, and they repeat association-dissociation with 24-hrs period as follows, A2+B4+C6 → A2C6+B4 → A2+B6C6 → A12B6C6 → A2+B4+C6 → ∙∙∙. To understand this system, it is essential to reveal the complex structures in every phase. For this purpose, we have been focused on the...
Neutron scattering experiments involving soft matter materials often require specific contrast to observe different parts of the materials. In order to increase the availability of deuterium labelled materials, we are establishing deuteration support to MLZ users. Proposals for the deuteration support can now be submitted in combination with proposals for neutron beamtime.
Our main...
KWS-2 represents a classical pinhole SANS diffractometer where, combining the conventional mode using different neutron wavelengths and detection distances with the focusing mode using MgF2 lenses, a wide Q-range between 1 x 10-4 and 1 Å-1 can be explored. The high neutron flux, comparable with that of the world leading SANS instruments, which is supplied by the neutron delivery system (cold...
The second target station (STS) is a >$1Billion, Department of Energy project to be constructed at Oak Ridge National Laboratories Spallation Neutron Source (SNS). The STS will provide entirely new capabilities for studying a broad range of materials with neutron scattering and support a wide variety of users. The science capabilities provided by the instrument suite at the STS will...
Currently, the scientific park of research instruments at the high flux pulsed reactor IBR-2 in FLNP JINR (Dubna, Moscow Region, Russia) consists of a complex of 15 neutron spectrometers for condensed matter studies, including 8 diffractometers, 3 reflectometers, 1 small-angle neutron scattering spectrometer, 2 inelastic neutron scattering spectrometers, and 1 spectrometer for neutron...
Candida glabrata has been known as a non-pathogenic yeast found in healthy humans, but the number of infections caused by it has increased, making understanding its virulence an urgent task. In our multidisciplinary effort, we combine methods to produce C. glabrata strains with well-defined genetic modifications of virulence/resistance factors(1) with characterization of their membrane...
Proteins are of enormous importance to life on earth. They have a multitude of different functions in all organisms and can work as enzymes, gene regulators, structural components, transporters, and receptors. Most drugs act on proteins. The structures and mechanisms of proteins are therefore prominent topics in life science research.
Access to both state-of-the-art X-ray (MAX IV) and...
Neutron single crystal diffraction provides an experimental method for the direct location of hydrogen and deuterium atoms in biological macromolecules, thus providing important complementary information to that gained by X-ray crystallography. At the FRM II neutron source in Garching near Munich the neutron single crystal diffractometer BIODIFF, a joint project of the Forschungszentrum Jülich...
Epithelial branch elongation is a central developmental process during branching morphogenesis in diverse organs. This fundamental growth process of large arborized epithelial networks is accompanied with huge structural reorganizations of the surrounding Extracellular Matrix (ECM), which is well beyond its mechanical linear response regime. Here, we report that epithelial ductal elongation...
Proteins are an important component in many medical and food products, and the long-time properties of these products are directly dependent on the stability of their proteins. To enhance this stability it has become common to add disaccharides in general, and trehalose in particular. However, the mechanisms by which disaccharides stabilize proteins and other biological materials are still not...
Many low molecular weight compounds and peptides are capable of forming supramolecular complexes. In the form of such complexes, the molecules are capable of multicenter cooperative binding to target proteins. It is advisable to study these complexes using small-angle scattering methods in combination with molecular dynamics modeling in the free diffusion approach.
When studying the mechanism...
The dynamical properties of lipid membranes play an important role in how cells, virus and organelles interact with the world around them. These properties arise from a complex interplay of forces over a wide range of time and length scales and, as such, are ideally suited for study with neutron spin echo (NSE) spectroscopy. Recently, the NSE technique has been extended to grazing incidence...
Proteome adaptation to high pressure in Archaea is still an open debate. Whole genome comparative studies could not identify a clear adaptation pattern, and HP adaptation is often considered as concomitant to another adaptation, for instance to high or low temperature.
Studies on whole cells of the near isogenic HP-adapted T. barophilus (T=85°C, p=400bars, piezophilic) and HP-sensitive *T....
Proteins are essential macronutrients in the human diet, being fundamental in body structure and functions. The protein digestibility depends not only on their composition but also on food structure, which in turn can be influenced by different types of processing.
We monitored degradation kinetics of the structure during simulated gastric and intestinal digestion, and analyzed its impact on...
Mucus is a highly viscoelastic secretion, covering the epithelia surfaces of several body tracts. Its function and composition differ at different locations, but its general task is to protect tissues from dehydration, mechanical stress, and to act as barrier against microorganisms and toxic substances. Mucus is mainly composed of water, lipids, small proteins and nucleic acids, but its...
Double hydrophilic block copolymers can self-assemble in water, leading to scaffolds with strong potential in favor of controlled encapsulation of pharmaceutics. We recently demonstrated that thermoresponsive double hydrophilic poly(N-isopropylacrylamide)-block-poly(oligo ethylene glycol methyl ether acrylate) (PNIPAM-b-POEGA) block copolymer self-assemblies in water can successfully...
Radioisotopes are an indispensable tool for diagnostics and therapy in nuclear medicine. As a rule of thumb, proton rich isotopes are most efficiently produced by accelerators, whereas neutron rich isotopes are more efficiently produced by neutron capture reactions. Here, we will focus on the latter process. Inherent to the purpose of nuclear medicine, most of these isotopes are short-lived...
Fluorescent proteins (FPs) have revolutionized the imaging technologies in biological science. A better understanding of the structure and function for FPs will help to develop new molecular designs to generate further practical devices. Recently, we reported neutron structural analysis of the green fluorescent protein (GFP) to show the characteristic protonation (deuteration) states of the...
COVID-19, caused by SARS-CoV-2, is a global health and economic catastrophe. The viral main protease (Mpro) is indispensable for SARS-CoV-2 replication and thus is an important target for small-molecule antivirals. Neutrons are an ideal probe to observe protonation states of ionizable amino acids at near-physiological temperature, directly determining their electric charges – crucial...
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is responsible for photosynthetic CO2 fixation and is the most abundant enzyme on earth. The carbon atoms in every organism in the entire biosphere have passed through the reaction cycle at the active site of a Rubisco enzyme at some point in time. In the photosynthetic CO2 reduction reaction catalysed by Rubisco, atmospheric CO2 is...
The elucidation of the protein-protein interaction, especially among membrane proteins and protein complexes, is one of the most important research fields in life science. Such proteins have large molecular weights, and the lattice lengths of their crystals have large values. Cold neutrons contribute to improve the difficulty in separating Bragg peaks from those crystals. JRR-3 has three cold...
Daniele Di Bari1,2,3, Stepan Timr4, Fabio Sterpone4, Alessandro Paciaroni1, Judith Peters2,3.
1Univ. of Perugia, Dep. of Physics and Geology, Italy. 2Univ. Grenoble-Alpes, CNRS, LiPhy, France. 3Institut Laue-Langevin, France. 4Laboratoire de Biochimie Theorique, CNRS, France.
Life on Earth...
The structure-dynamics-function relationship in proteins remains a field of great scientific interest. Photoactive proteins form a specific class, whose function can be activated by illumination. Two prototypical examples are the Orange Carotenoid Protein (OCP) and bacteriorhodopsin (BR). As to the first, photodamage of the photosynthetic apparatus of cyanobacteria in the case of excess light...
Triosephosphate isomerase (TIM) is a key enzyme in glycolysis that catalyses the interconversion of glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). This simple reaction involves the shuttling of protons mediated by protolysable side chains. The catalytic power of TIM is thought to stem from the ability to facilitate the deprotonation of a carbon next to a carbonyl group...
We have used the ability of neutron crystallography to locate hydrogen atoms in our studies to investigate the mechanisms of the heme peroxidases cytochrome c peroxidase and ascorbate peroxidase. In order to do this we have cryo-trapped the labile intermediates known as Compound I and Compound II in crystals. A key question about these intermediates has been the protonation states of the...
The fourth enzymatic reaction in the de novo pyrimidine biosynthesis, the oxidation of dihydroorotate to orotate, is catalyzed by dihydroorotate dehydrogenase (DHODH). Enzymes belonging to the DHODH Class II are membrane-bound proteins that use ubiquinone as their electron acceptor. We designed this study to understand the interaction of an N-terminally truncated version of human DHODH...
Polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA) is found in very high concentrations in a few peculiar tissues. DHA was proposed to affect the function of the cell membrane and related proteins through an indirect mechanism of action, based on the DHA-phospholipid effects on the bilayer structure. Most studies have focused on its influence on lipid-rafts, neglecting the effects...
Lactoferrin (Lf) is a non-heme protein known for its ability to naturally bind tightly Fe3+ ions in various physiological environments. Therefore, Lf has a significant role in the processes of iron regulation at the cellular level and organize the specific and non-specific immune response in the body.
The conformational changes within the protein structure caused by the iron-binding are...
The protein-protein interactions, namely those between the cationic lactoferrin and the oppositely charged β-lactoglobulin have been the subject of many studies due to their appeal for different food and pharmaceutical applications. These two proteins show some peculiarities in terms of physicochemical properties and behaviour in solutions. Although the hetero-complexation of the proteins is...
Phytochromes are red/far-red photochromic biliprotein light sensors that act as master regulators of plant development. They regulate the expression of ~20% of all plant genes, controlling for example germination, pigment production, stem extension and flowering time. Phytochromes are also known in prokaryotes, Cph1 from Synechocystis, for example, providing a valuable model for...
Despite of the pivotal role that hydrogen (H) atoms play in protein biological function, and the fact these comprise approximately 50% of all protein atoms, their observation through X-ray diffraction remains elusive. Conversely, neutron diffraction data at resolutions better than 2.5 Å allows the determination of H positions, providing unique insight to the catalytic mechanisms of enzymes....
After an introduction into strategies for controlling the time-averaged behavior of aqueous protein solutions in the bulk at near interfaces, we discuss their dynamical behavior.
First, this concerns the impact of temperature, salt concentration, protein concentration and other control parameters on the diffusion in equilibrium.
Second, we study how the dynamics in these complex systems...
Knowledge of the protein tracer diffusion constitutes a key element to describe intracellular transport, which can be modeled by the self-diffusion in colloid systems. However, it is necessary to test the underlying assumption that neither the protein shape and size nor the polydisperse nature of the cytosol matter. We present a combined experimental and simulation study of the protein tracer...
Infrared spectroscopy serves as local probe reporting on specific vibrations in some side chains which are spectrally distant from the complicated infrared spectrum of a protein in solution. Here, infrared spectroscopy can give information on the fold of the protein and also follow aggregation phenomena. Small angle neutron scattering also reports on the global structure of proteins in...
PcyA reduces biliverdin IXα (BV), a heme degradation product, in a ferredoxin-dependent manner, to synthesize phycocyanobilin, which plays an important role in photosynthesis and biological photoresponse. PcyA is a unique enzyme that sequentially reduces D-ring vinyl group and A-ring vinyl group of BV in a site-specific manner. In this study, Ile86 located near the important amino acid Asp105,...
The investigation of fiber distribution and myelin orientation in the brain has received increasing attention in recent years, as the 3D fiber structure reveals the connectivity of the axonal network (connectome) that is necessary to understand dysfunctions of the brain [1]. Hence, we adapt scanning small angle neutron/x-ray scattering (sSANS/sSAXS) to map an entire brain section of a mouse...
Severe acute respiratory syndrome (SARS) is a viral infectious disease caused by the new coronavirus strain (CoV2). The SARS-CoV2 replication and transcription complex (RTC) is formed with at least 9 NSPs that are arranged into one functional assembly. The non-structural proteins (NSPs) Nsp7 and 8 are important components of this complex. Our overall aim was to investigate the structural...
Upon protein ligand binding, changes of conformational entropy occur in protein and hydration layer. In an experimental study, we investigated the relevance of conformational entropy for the binding of biotin to the protein streptavidin. Using QENS, we investigated changes in conformational entropy between the ligand-free and the ligand-bound state. We compared the internal dynamics of...
Therapeutic treatments based on the production of proteins by delivering messenger RNA (mRNA) represent a versatile approach. Lipid nanoparticles (LNPs) are promising vehicles for mRNA delivery and are formed by a cationic ionizable lipid (CIL), DSPC, cholesterol (Chol) and a pegylated (PEG) lipid. Even though some LNPs for small interference RNA (siRNA) delivery were recently FDA approved,...
Crystallography and cryo-EM have provided new and exciting insight into protein substrate degradation by AAA+ ATPases and the PAN-proteasome system. However, direct structural information on the conformational changes of the working complex, as well as the respective substrate states and populations during the active unfolding and degradation process remain scarce.
Here, we apply...
Lipid nanoparticles (LNPs) constituted by a cationic ionizable lipid and helper lipids as cholesterol, phospholipids and poly(ethylene glycol) lipid as stabilizer have made mRNA therapeutics a reality. 2 mRNA-based vaccines against SARS-CoV-2 have received emergency authorization by many regulatory agencies using LNPs as delivery vehicle. We investigated the structure of mRNA-containing LNPs...
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) papain-like protease (PLpro) is essential for the virus replication. PLpro has the additional function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. PLpro is thus an excellent drug target for a two-fold strategy to...
Two influenza A nucleoprotein variants (wt: G102R; and mutant: G102R and E292G) were studied with regard to macro-molecular interactions in oligomeric form (24-mers). The E292G mutation has been previously shown to provide cold adaptation. Molecular dynamics simulations of these complexes and trajectory analysis showed that the most significant difference between the obtained models was...
Structural changes at the intra- and interchain level induced by the phase transition of poly(N-isopropylacryl amide) (PNIPAM) can be tracked in real-time by time-resolved small-angle neutron scattering (tr-SANS). PNIPAM is one of the most commonly and extensively studied thermoresponsive polymer due to lower critical solution temperature (LCST) in water that occurs at the physiologically...
We have investigated the impact of the drugs benzocaine and propanolol on a lipid bilayer formed by L-alpha-phosphatidylcholine. The methods used were neutron reflectivity, grazing incidence small angle neutron scattering, small and ultra small angle neutron scattering. On the one hand, we observed a membrane stiffening and a stalk formation for benzocaine. On the other hand, disordered...
KRas4B is a membrane-anchored signaling protein and primary target in cancer research. Predictions from molecular dynamics simulations have previously shaped our mechanistic understanding of KRas signaling but disagree with recent experimental results from neutron reflectometry, nuclear magnetic resonance, and thermodynamic binding studies [1]. We compare this body of biophysical data to...
Despite all advances in cancer treatment, there are still clinical situations in which a locoregional tumor is fatal. More efficient local therapies are mandatory. Boron Neutron Capture Therapy (BNCT) has the potential of such a modality to overcome radiation resistance in certain tumors or to re-irradiate local recurrences after high-dose radiotherapy.
BNCT is based on the 10B(n,α)7Li...
The outbreak of the coronavirus disease (COVID-19) caused by the coronavirus SARS-CoV-2 spread to every continent affecting the global health and economy. The first two open reading frame of the SARS-CoV-2 genome are translated into two polyproteins.These are cleaved into 16 non-structural proteins (nsp1-nsp16), which are essential for viral replication and transcription [1].Among these,...
The major iron uptake mechanism in bacteria is mediated by ABC transporters of the FutA type. We study iron uptake and homeostasis in highly adapted marine cyano-bacteria that are of high geochemical significance for global net carbon fixation. We study FutA substrate binding domains that bind iron in the Fe(III) oxidation state. FutA proteins also have an intracellular function in protection...
