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Naturally occurring plant cellulose, our most abundant renewable resource, consists of fibers of long polymer chains that are tightly packed in parallel arrays in either of two crystal phases collectively referred to as cellulose I. During mercerization, a process that involves treatment with sodium hydroxide, cellulose goes through a conversion to another crystal form called cellulose II, within which every other chain has remarkably changed direction. We designed a neutron diffraction experiment with deuterium labelling in order to understand how this change of cellulose chain direction is possible. Here we show that during mercerization of bacterial cellulose, chains fold back on themselves in a zigzag pattern to form crystalline anti-parallel domains. This result provides a molecular level understanding of one of the most widely used industrial processes for improving cellulosic materials., (© 2022. Crown.)

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. Distinct folding modes are observed for the hydrogenated and perdeuterated refolded variants, which are determined by conformational changes to the backbone structure of the Lys97-Gly104 flexible loop. Surprisingly, the structure of the refolded perdeuterated protein is closer to that of native lysozyme than that of the refolded hydrogenated protein. These structural differences suggest that the observed decreases in thermal stability and enzymatic activity in the refolded perdeuterated and hydrogenated proteins are consequences of the macromolecular deuteration effect and of distinct folding dynamics, respectively. These results are discussed in the context of both in vitro and in vivo folding, as well as of lysozyme amyloidogenesis., (open access.)

Crystals of Cs 6 (SO 4 ) 3 (H 3 PO 4 ) 4 belong to the family of alkali metal acid salts that show a high protonic conductivity at relatively low temperatures. Such properties make superprotonic crystals an excellent choice for the study of the influence of the hydrogen subsystem on the physicochemical properties and promising materials for energy-efficient technologies. Single crystals of Cs 6 (SO 4 ) 3 (H 3 PO 4 ) 4 were studied by neutron diffraction methods, optical polarization microscopy, scanning electron microscopy and energy-dispersive X-ray spectroscopy. Neutron diffraction studies made it possible to determine the positions of all the atoms with high accuracy, including the H atom on a hydrogen bond characterized by a single-minimum potential, to confirm the chemical composition of the Cs 6 (SO 4 ) 3 (H 3 PO 4 ) 4 crystals and their cubic symmetry in low- and high-temperature phases, and to draw conclusions about the three-dimensional system of hydrogen bonds, which is fundamentally different in comparison with other superprotonic compounds. Based on the experimental data obtained, crystal transformations with temperature changes are reported, and the stability of the chemical composition is shown.

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., (© Ramos et al. 2021.)

The melting transition of Li-DNA fibers immersed in ethanol-water solutions has been studied using calorimetry and neutron diffraction techniques. The data have been analyzed using the Peyrard-Bishop-Dauxois model to determine the strengths of the intra- and inter-base pair potentials. The data and analysis show that the potentials are weaker than those for DNA in water. They become weaker still and the DNA less stable as the ethanol concentration increases but, conversely, the fibers become more compact and the distances between base pairs become more regular. The results show that the melting transition is relatively insensitive to local confinement and depends more on the interaction between the DNA and its aqueous environment., (© 2021 Wiley Periodicals LLC.)

Deprotonation usually occurs as an unwanted side reaction in the Lewis pair polymerization of Michael acceptors, for which the conjugated addition of the Lewis base to the acid-activated monomer is the commonly accepted initiation mechanism. This has also been reported for B-P-based bridged Lewis pairs (BLPs) that form macrocyclic addition products. We now show that the formerly unwanted deprotonation is the likely initiation pathway in the case of Al-P-based BLPs. In a detailed study of a series of Al-P-based BLPs, using a combination of single-crystal diffraction experiments (X-ray and neutron) and mechanistic investigations (experimental and computational), an active role of the methylene bridge was revealed, acting as a base towards the α-acidic monomers. Additionally, the polymerization studies proved a living behavior combined with significantly high activities, narrow molecular mass distributions, and the possibility of copolymerization., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe a novel self-assembling supramolecular nanotube system formed by a heterocyclic cationic molecule which was originally designed for its potential as an antiparasitic and DNA sequence recognition agent. Our structural characterisation work indicates that the nanotubes form via a hierarchical assembly mechanism that can be triggered and tuned by well-defined concentrations of simple alkali halide salts in water. The nanotubes assembled in NaCl have inner and outer diameters of ca. 22 nm and 26 nm respectively, with lengths that reach into several microns. Our results suggest the tubes consist of DB921 molecules stacked along the direction of the nanotube long axis. The tubes are stabilised by face-to-face π-π stacking and ionic interactions between the charged amidinium groups of the ligand and the negative halide ions. The assembly process of the nanotubes was followed using small-angle X-ray and neutron scattering, transmission electron microscopy and ultraviolet/visible spectroscopy. Our data demonstrate that assembly occurs through the formation of intermediate ribbon-like structures that in turn form helices that tighten and compact to form the final stable filament. This assembly process was tested using different alkali-metal salts, showing a strong preference for chloride or bromide anions and with little dependency on the type of cation. Our data further demonstrates the existence of a critical anion concentration above which the rate of self-assembly is greatly enhanced.

The influence of molecular confinement on the melting transition of oriented Na-DNA fibers submerged in poly(ethylene glycol) (PEG) solutions has been studied. The PEG solution exerts an osmotic pressure on the fibers which, in turn, is related to the DNA intermolecular distance. Calorimetry measurements show that the melting temperature increases and the width of the transition decreases with decreasing intermolecular distance. Neutron scattering was used to monitor the integrated intensity and width of a Bragg peak from the B-form of DNA as a function of temperature. The data were quantitatively analyzed using the Peyrard-Bishop-Dauxois model. The experiments and analysis showed that long segments of double-stranded DNA persist until the last stages of melting and that there appears to be a substantial increase of the DNA dynamics as the melting temperature of the DNA is approached.

A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked β-strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual α-synuclein amyloids., (© 2017 The Authors Cytoskeleton Published by Wiley Periodicals, Inc.)

Serial diffraction data collected at the Linac Coherent Light Source from crystalline amyloid fibrils delivered in a liquid jet show that the fibrils are well oriented in the jet. At low fibril concentrations, diffraction patterns are recorded from single fibrils; these patterns are weak and contain only a few reflections. Methods are developed for determining the orientation of patterns in reciprocal space and merging them in three dimensions. This allows the individual structure amplitudes to be calculated, thus overcoming the limitations of orientation and cylindrical averaging in conventional fibre diffraction analysis. The advantages of this technique should allow structural studies of fibrous systems in biology that are inaccessible using existing techniques.

In this study, we propose a photostructuring approach for protein films based on a treatment with nanosecond pulses of a KrF excimer laser. As a model protein we used an amyloid fibril-forming protein. Laser treatment induced a foaming of the sample surface exhibiting an interconnected fibrous mesh with a high degree of control and precision. The surface foaming was well characterized by scanning electron microscopy, atomic force microscopy, laser induced fluorescence and contact angle measurements. The laser irradiated areas of the protein films acquired new morphological and physicochemical properties that could be exploited to fulfill unmet challenges in the tissue engineering field. In this context we subsequently evaluated the response of NIH/3T3 fibroblast cell line on the processed film. Our results show a strong and statistically significant preference for adhesion and proliferation of cells on the irradiated areas compared to the non-irradiated ones. We propose that this strategy can be followed to induce selective cell patterning on protein films at the microscale.

Self-assembled peptides gain increasing interest as biocompatible and biodegradable scaffolds for tissue engineering. Rationally designed self-assembling building blocks that carry cell adhesion motifs such as Arg-Gly-Asp (RGD) are especially attractive. We have used a combination of theoretical and experimental approaches toward such rational designs, especially focusing on modular designs that consist of a central ultrashort amphiphilic motif derived from the adenovirus fiber shaft. In this study, we rationally designed RGDSGAITIGC, a bifunctional self-assembling amyloid peptide which encompasses cell adhesion and potential cysteine-mediated functionalization properties through the incorporation of an RGD sequence motif and a cysteine residue at the N- and C- terminal end, respectively. We performed replica exchange MD simulations that suggested that the key factor determining cell adhesion is the total solvent accessibility of the RGD motif and also that the C-terminal cysteine is adequately exposed. The designer peptides self-assembled into fibers that are structurally characterized with Transmission Electron Microscopy, Scanning Electron Microscopy and X-ray fiber diffraction. Furthermore, they supported cell adhesion and proliferation of a model cell line. We consider that the current bifunctional properties of the RGDSGAITIGC fibril-forming peptide can be exploited to fabricate novel biomaterials with promising biomedical applications. Such short self-assembling peptides that are amenable to computational design offer open-ended possibilities toward multifunctional tissue engineering scaffolds of the future.

Neutron diffraction of tri(3,5-tert-butylphenyl)methane at 20 K reveals an intermolecular C-H···H-C distance of only 1.566(5) Å, which is the shortest reported to date. The compound crystallizes as a C 3 -symmetric dimer in an unusual head-to-head fashion. Quantum chemical computations of the solid state at the HSE-3c level of theory reproduce the structure and the close contact well (1.555 Å at 0 K) and emphasize the significance of packing effects; the gas-phase dimer structure at the same level shows a 1.634 Å C-H···H-C distance. Intermolecular London dispersion interactions between contacting tert-butyl substituents surrounding the central contact deliver the decisive energetic contributions to enable this remarkable bonding situation.

In this report we show for the first time that neutron anomalous dispersion can be used in a practical manner to determine experimental phases of a protein crystal structure, providing a new tool for structural biologists. The approach is demonstrated through the use of a state-of-the-art monochromatic neutron diffractometer at the Institut Laue-Langevin (ILL) in combination with crystals of perdeuterated protein that minimise the level of hydrogen incoherent scattering and enhance the visibility of the anomalous signal. The protein used was rubredoxin in which cadmium replaced the iron at the iron-sulphur site. While this study was carried out using a steady-state neutron beam source, the results will be of major interest for capabilities at existing and emerging spallation neutron sources where time-of-flight instruments provide inherent energy discrimination. In particular this capability may be expected to offer unique opportunities to a rapidly developing structural biology community where there is increasing interest in the identification of protonation states, protein/water interactions and protein-ligand interactions - all of which are of central importance to a wide range of fundamental and applied areas in the biosciences.

The paired helical filaments (PHF) formed by the intrinsically disordered human protein tau are one of the pathological hallmarks of Alzheimer disease. PHF are fibers of amyloid nature that are composed of a rigid core and an unstructured fuzzy coat. The mechanisms of fiber formation, in particular the role that hydration water might play, remain poorly understood. We combined protein deuteration, neutron scattering, and all-atom molecular dynamics simulations to study the dynamics of hydration water at the surface of fibers formed by the full-length human protein htau40. In comparison with monomeric tau, hydration water on the surface of tau fibers is more mobile, as evidenced by an increased fraction of translationally diffusing water molecules, a higher diffusion coefficient, and increased mean-squared displacements in neutron scattering experiments. Fibers formed by the hexapeptide (306)VQIVYK(311) were taken as a model for the tau fiber core and studied by molecular dynamics simulations, revealing that hydration water dynamics around the core domain is significantly reduced after fiber formation. Thus, an increase in water dynamics around the fuzzy coat is proposed to be at the origin of the experimentally observed increase in hydration water dynamics around the entire tau fiber. The observed increase in hydration water dynamics is suggested to promote fiber formation through entropic effects. Detection of the enhanced hydration water mobility around tau fibers is conjectured to potentially contribute to the early diagnosis of Alzheimer patients by diffusion MRI.

The two main branches of bionanotechnology involve the self-assembly of either peptides or DNA. Peptide scaffolds offer chemical versatility, architectural flexibility and structural complexity, but they lack the precise base pairing and molecular recognition available with nucleic acid assemblies. Here, inspired by the ability of aromatic dipeptides to form ordered nanostructures with unique physical properties, we explore the assembly of peptide nucleic acids (PNAs), which are short DNA mimics that have an amide backbone. All 16 combinations of the very short di-PNA building blocks were synthesized and assayed for their ability to self-associate. Only three guanine-containing di-PNAs-CG, GC and GG-could form ordered assemblies, as observed by electron microscopy, and these di-PNAs efficiently assembled into discrete architectures within a few minutes. The X-ray crystal structure of the GC di-PNA showed the occurrence of both stacking interactions and Watson-Crick base pairing. The assemblies were also found to exhibit optical properties including voltage-dependent electroluminescence and wide-range excitation-dependent fluorescence in the visible region.

A novel vitreous carbon mount for macromolecular crystallography, suitable for neutron and X-ray crystallographic studies, has been developed. The technology described here is compatible both with X-ray and neutron cryo-crystallography. The mounts have low density and low background scattering for both neutrons and X-rays. They are prepared by laser cutting, allowing high standards of production quality, the ability to custom-design the mount to specific crystal sizes and large-scale production.

The title zwitterion (2S)-2-azaniumyl-1-hydroxy-3-phenylpropan-1-olate, C9H11NO2, also known as L-phenylalanine, was characterized using synchrotron X-rays. It crystallized in the monoclinic space group P21 with four molecules in the asymmetric unit. The 0.62 Å resolution structure is assumed to be closely related to the fibrillar form of phenylalanine, as observed by electron microscopy and electron diffraction. The structure exists in a zwitterionic form in which π-π stacking and hydrogen-bonding interactions are believed to form the basis of the self-assembling properties.

In this study, we propose a new approach to hard tissue regeneration based on the mineralization of 3D scaffolds made using lasers. To this end, we report the rational design of aspartate-containing self-assembling peptides targeted for calcium binding. We further investigate the suitability of these peptides to support cell attachment and proliferation when coupled on a hybrid organic-inorganic structurable material, and evaluate the response of pre-osteoblastic cells on functionalized 3D scaffolds and material surfaces. Our results show that the mineralized peptide, when immobilized on a hybrid photo-structurable material strongly supports cell adhesion, a proliferation increase after three and seven days in culture, and exhibits a statistically significant increase of biomineralization. We propose this strategy as a 'scaffold on scaffold' approach for hard tissue regeneration.

Biocompatible hydrogels are of high interest as a class of biomaterials for tissue engineering, regenerative medicine, and controlled drug delivery. These materials offer three-dimensional scaffolds to support the growth of cells and development of hierarchical tissue structures. Fmoc-peptides were previously demonstrated as attractive building blocks for biocompatible hydrogels. Here, we further investigate the biophysical properties of Fmoc-peptide-based hydrogels for medical applications. We describe the structural and thermal properties of these Fmoc-peptides, as well as their self-assembly process. Additionally, we study the role of interactions between aromatic moieties in the self-assembly process and on the physical and structural properties of the hydrogels., (© 2012 American Chemical Society)

The increase in the number of large multi-component complexes and membrane protein crystal structures determined over the last few years can be ascribed to a number of factors such as better protein expression and purification systems, the emergence of high-throughput crystallization techniques and the advent of 3rd generation synchrotron sources. However, many systems tend to produce crystals that can be extremely heterogeneous in their diffraction properties. This prevents, in many cases, the collection of diffraction data of sufficient quality to yield useful biological or phase information. Techniques that can increase the diffraction quality of macromolecular crystals can therefore be essential in the successful conclusion of these challenging projects. No technique is universal but encouraging results have been recently achieved by carrying out the controlled dehydration of crystals of biological macromolecules. A new device that delivers a stream of air with a precisely controlled relative humidity to the complicated sample environment found at modern synchrotron beamlines has been conceived at the EMBL Grenoble and developed by the EMBL and the ESRF as part of the SPINE2 complexes project, a European Commission funded protein structure initiative. The device, the HC1b, has been available for three years at the ESRF macromolecular crystallography beamlines and many systems have benefitted from on-line controlled dehydration. Here we describe a standard dehydration experiment, highlight some successful cases and discuss the different possible uses of the device., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The first neutron fibre diffraction studies of an amyloid system are presented. The techniques used to prepare the large samples needed are described, as well as the procedures used to isotopically replace H2O in the sample by D2O. The results demonstrate the feasibility of this type of approach for the pursuit of novel structural analyses that will strongly complement X-ray fibre diffraction studies and probe aspects of amyloid structure that to date have remained obscure. The approach is demonstrated using an amyloid form of the peptide NSGAITIG, but is equally applicable for the study of other systems such as Alzheimer's Aβ peptide.

The ability to develop a rational basis for the binding of inorganic materials to specific binding sites within self-assembling biological scaffolds has important applications in nanobiotechnology. Amyloid-forming peptides are a class of such scaffolds and show enormous potential as templates for the fabrication of low resistance, conducting nanowires. Here we report the use of a self-assembling peptide building block as scaffold for the systematic introduction of metal-binding residues at specific locations within the structure. The octapeptide NSGAITIG (Asparagine-Serine-Glycine-Alanine-Isoleucine-Threonine-Isoleucine-Glycine) from the fiber protein of adenovirus has been identified in previous structural studies as an elementary fibril-forming building block. Using this building block as a scaffold, we have designed three new cysteine-containing octa-peptides to study their eventual fibril-forming ability and potential templating of metal nanoparticles. We find that the cysteine substitutions do not alter the fibril-forming potential of the peptides, and that the fibrils formed bind efficiently to silver, gold, and platinum nanoparticles; furthermore, we report unexpected behavior of serine in nucleating gold and platinum nanoparticles. We find that combination of cysteine and serine residues projecting from adjacent sites on a peptide scaffold represents a potentially useful strategy in nucleating inorganic materials. The ability to reliably produce metal-coated fibrils is a vital first step towards the exploitation of these fibrils as conducting nanowires with applications in nano-circuitry. Short, biologically inspired self-assembling peptide scaffolds derived from natural fibrous proteins with known three-dimensional structure may provide a viable approach towards the rational design of inorganic nanowires., ((c) 2009 Wiley Periodicals, Inc.)