Two populations of protein molecules detected by small-angle neutron and X-ray scattering (SANS and SAXS) in lyophilized protein:lyoprotector (disaccharide) systems.

Two protein interaction peaks are observed in pharmaceutically-relevant protein (serum albumin) : disaccharide 1 : 1 and 1 : 3 (w/w) freeze-dried systems for the first time. In samples with a higher disaccharide content, the protein-protein distances are longer for both populations, while the fraction of the protein population with a shorter protein-protein distance is lower. Both factors would favor better stability against aggregation for disaccharide-rich protein formulations. This study provides direct experimental support for a "dilution" hypothesis as a potential stabilization mechanism for freeze-dried protein formulations.

Protein-ligand interactions from a quantum fragmentation perspective: The case of the SARS-CoV-2 main protease interacting with α-ketoamide inhibitors.

We present a hybrid, multi-method, computational scheme for protein/ligand systems well suited to be used on modern and forthcoming massively parallel computing systems. The scheme relies on a multi-scale polarizable molecular modeling, approach to perform molecular dynamics simulations, and on an efficient Density Functional Theory (DFT) linear scaling method to post-process simulation snapshots. We use this scheme to investigate recent α-ketoamide inhibitors targeting the main protease of the SARS-CoV-2 virus. We assessed the reliability and the coherence of the hybrid scheme, in particular, by checking the ability of MM and DFT to reproduce results from high-end ab initio computations regarding such inhibitors. The DFT approach enables an a posteriori fragmentation of the system and an investigation into the strength of interaction among identified fragment pairs. We show the necessity of accounting for a large set of plausible protease/inhibitor conformations to generate reliable interaction data. Finally, we point out ways to further improve α-ketoamide inhibitors to more strongly interact with particular protease domains neighboring the active site.

Bowtie-Shaped Deformation Isotherm of Superhydrophobic Cylindrical Mesopores.

Deformation of superhydrophobic cylindrical mesopores is studied during a cycle of forced water filling and spontaneous drying by in situ small-angle neutron scattering. A high-pressure setup is put forward to characterize the deformation of ordered mesoporous silanized silica up to 80 MPa. Strain isotherms of individual pores are deduced from the shift of the Bragg spectrum associated with the deformation of the hexagonal pore lattice. Due to their superhydrophobic nature, pore walls are not covered with a prewetting film. This peculiarity gives the ability to use a simple mechanical model to describe both filled and empty pore states without the pitfall of disjoining pressure effects. By fitting our experimental data with this model, we measure both the Young's modulus and the Poisson ratio of the nanometric silica wall. The measurement of this latter parameter constitutes a specificity offered by superhydrophobic nanopores with respect to hydrophilic ones.

Zinc determines dynamical properties and aggregation kinetics of human insulin.

Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment, and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here, we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, in which the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced x-ray emission and x-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.

Melting transition of oriented Li-DNA fibers submerged in ethanol solutions.

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.

Primary and Secondary Hydration Forces between Interdigitated Membranes Composed of Bolaform Microbial Glucolipids.

To better understand lipid membranes in living organisms, the study of intermolecular forces using the osmotic pressure technique applied to model lipid membranes has constituted the ground knowledge in the field of biophysics since four decades. However, the study of intermolecular forces in lipid systems other than phospholipids, like glycolipids, has gained a certain interest only recently. Even in this case, the work generally focuses on the study of membrane glycolipids, but little is known on new forms of non-membrane functional compounds, like microbial bolaform glycolipids. This work explores, through the osmotic stress method involving an adiabatic humidity chamber coupled to neutron diffraction, the short-range (<2 nm) intermolecular forces of membranes entirely composed of interdigitated glucolipids. Experiments are performed at pH 6 when the glucolipid is partially negatively charged and for which we explore the effect of low (16 mM) and high (100 mM) ionic strength. We find that this system is characterized by primary and secondary hydration regimes insensitive and sensitive to ionic strength, respectively, and with typical decay lengths of λH1 = 0.37 ± 0.12 nm and λH2 = 1.97 ± 0.78 nm.

Anesthetics significantly increase the amount of intramembrane water in lipid membranes.

The potency of anesthesia was directly linked to the partitioning of the drug molecules in cell membranes by Meyer and Overton. Many molecules interact with lipid bilayers and lead to structural and functional changes. It remains an open question which change in membrane properties is responsible for a potential anesthetic effect or if anesthetics act by binding to direct targets. We studied the effect of ethanol, diethyl ether and isoflurane on the water distribution in lipid bilayers by combining all-atom molecular dynamics simulations and neutron diffraction experiments. The simulations show strong membrane-drug interactions with partitioning coefficients of 38%, 92% and 100% for ethanol, diethyl ether and isoflurane, respectively, and provide evidence for an increased water partitioning in the membrane core. The amount of intramembrane water molecules was experimentally determined by selectively deuterium labeling lipids, anesthetic drug and water molecules in neutron diffraction experiments. Four additional water molecules per lipid were observed in the presence of ethanol. Diethyl ether and isoflurane were found to significantly increase the amount of intramembrane water by 25% (8 water molecules). This increase in intramembrane water may contribute to the non-specific interactions between anesthetics and lipid membranes.

Flexibilities of wavelets as a computational basis set for large-scale electronic structure calculations.

The BigDFT project was started in 2005 with the aim of testing the advantages of using a Daubechies wavelet basis set for Kohn-Sham (KS) density functional theory (DFT) with pseudopotentials. This project led to the creation of the BigDFT code, which employs a computational approach with optimal features of flexibility, performance, and precision of the results. In particular, the employed formalism has enabled the implementation of an algorithm able to tackle DFT calculations of large systems, up to many thousands of atoms, with a computational effort that scales linearly with the number of atoms. In this work, we recall some of the features that have been made possible by the peculiar properties of Daubechies wavelets. In particular, we focus our attention on the usage of DFT for large-scale systems. We show how the localized description of the KS problem, emerging from the features of the basis set, is helpful in providing a simplified description of large-scale electronic structure calculations. We provide some examples on how such a simplified description can be employed, and we consider, among the case-studies, the SARS-CoV-2 main protease.

Bio-based glyco-bolaamphiphile forms a temperature-responsive hydrogel with tunable elastic properties.

A bio-based glycolipid bolaamphiphile (glyco-bolaamphiphile) has recently been produced (Van Renterghem et al., Biotechnol. Bioeng., 2018, 115, 1195-1206) on a gram scale by using the genetically-engineered S. bombicola strain Δat Δsble Δfao1. The glyco-bolaamphiphile bears two symmetrical sophorose headgroups at the extremities of a C16:0 (ω-1 hydroxylated palmitic alcohol) spacer. Its atypical structure has been obtained by redesigning the S. bombicola strain Δat Δsble, producing non-symmetrical glyco-bolaamphiphile, with an additional knock out (Δfao1) and feeding this new strain with fatty alcohols. The molecular structure of the glyco-bolaamphiphile is obtained by feeding the new strain a saturated C16 substrate (palmitic alcohol), which enables the biosynthesis of bolaform glycolipids. In this work, we show that the bio-based glyco-bolaamphiphile readily forms a hydrogel in water at room temperature, and that the hydrogel formation depends on the formation of self-assembled fibers. Above 28 °C, the molecules undergo a gel-to-sol transition, which is due to a fiber-to-micelle phase change. We provide a quantitative description of the Self-Assembled Fibrillar Network (SAFiN) hydrogel formed by the glyco-bolaampiphile. We identify the sol-gel transition temperature, the gelling time, and the minimal gel concentration; additionally, we explore the fibrillation mechanism as a function of time and temperature and determine the activation energy of the micelle-to-fiber phase transition. These parameters allow control of the elastic properties of the glyco-bolaamphiphile hydrogel: at 3 wt% and 25 °C, the elastic modulus G' is above the kPa range, while at 5 °C, G' can be tuned between 100 Pa and 20 kPa, by controlling the undercooling protocol.

Efficient internalization of TAT peptide in zwitterionic DOPC phospholipid membrane revealed by neutron diffraction.

The aim of this study is to investigate the interactions between TAT peptides and a neutral DOPC bilayer by using neutron lamellar diffraction. The distribution of TAT peptides and the perturbation of water distribution across the DOPC bilayer were revealed. When compared to our previous study on an anionic DOPC/DOPS bilayer (X. Chen et al., Biochim Biophys Acta. 2013. 1828 (8), 1982-1988), a much deeper insertion of TAT peptides was found in the hydrophobic core of DOPC bilayer at a depth of 6.0Å from the center of the bilayer, a position close to the double bond of fatty acyl chain. We conclude that the electrostatic attractions between the positively charged TAT peptides and the negatively charged headgroups of phospholipid are not essential for the direct translocation. Furthermore, the interactions of TAT peptides with the DOPC bilayer were found to vary in a concentration-dependent manner. A limited number of peptides first associate with the phosphate moieties on the lipid headgroups by using the guanidinium ions pairing. Then the energetically favorable water defect structures are adopted to maintain the arginine residues hydrated by drawing water molecules and lipid headgroups into the bilayer core. Such bilayer deformations consequently lead to the deep intercalation of TAT peptides into the bilayer core. Once a threshold concentration of TAT peptide in the bilayer is reached, a significant rearrangement of bilayer will happen and steady-state water pores will form.

Combination of acoustic levitation with small angle scattering techniques and synchrotron radiation circular dichroism. Application to the study of protein solutions.

BACKGROUND: The acoustic levitation technique is a useful sample handling method for small solid and liquids samples, suspended in air by means of an ultrasonic field. This method was previously used at synchrotron sources for studying pharmaceutical liquids and protein solutions using x-ray diffraction and small angle x-ray scattering (SAXS). METHODS: In this work we combined for the first time this containerless method with small angle neutron scattering (SANS) and synchrotron radiation circular dichroism (SRCD) to study the structural behavior of proteins in solutions during the water evaporation. SANS results are also compared with SAXS experiments. RESULTS: The aggregation behavior of 45µl droplets of lysozyme protein diluted in water was followed during the continuous increase of the sample concentration by evaporating the solvent. The evaporation kinetics was followed at different drying stage by SANS and SAXS with a good data quality. In a prospective work using SRCD, we also studied the evolution of the secondary structure of the myoglobin protein in water solution in the same evaporation conditions. CONCLUSIONS: Acoustic levitation was applied for the first time with SANS and the high performances of the used neutron instruments made it possible to monitor fast container-less reactions in situ. A preliminary work using SRCD shows the potentiality of its combination with acoustic levitation for studying the evolution of the protein structure with time. GENERAL SIGNIFICANCE: This multi-techniques approach could give novel insights into crystallization and self-assembly phenomena of biological compound with promising potential applications in pharmaceutical, food and cosmetics industry. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Claudin-11 Tight Junctions in Myelin Are a Barrier to Diffusion and Lack Strong Adhesive Properties.

The radial component is a network of interlamellar tight junctions (TJs) unique to central nervous system myelin. Ablation of claudin-11, a TJ protein, results in the absence of the radial component and compromises the passive electrical properties of myelin. Although TJs are known to regulate paracellular diffusion, this barrier function has not been directly demonstrated for the radial component, and some evidence suggests that the radial component may also mediate adhesion between myelin membranes. To investigate the physical properties of claudin-11 TJs, we compared fresh, unfixed Claudin 11-null and control nerves using x-ray and neutron diffraction. In Claudin 11-null tissue, we detected no changes in myelin structure, stability, or membrane interactions, which argues against the notion that myelin TJs exhibit significant adhesive properties. Moreover, our osmotic stressing and D2O-H2O exchange experiments demonstrate that myelin lacking claudin-11 is more permeable to water and small osmolytes. Thus, our data indicate that the radial component serves primarily as a diffusion barrier and elucidate the mechanism by which TJs govern myelin function.

Direct Comparison of Disaccharide Interaction with Lipid Membranes at Reduced Hydrations.

Understanding sugar-lipid interactions during desiccation and freezing is an important step in the elucidation of cryo- and anhydro-protection mechanisms. We determine sucrose, trehalose, and water concentration distributions in intra-bilayer volumes between opposing dioleoylphosphatidylcholine bilayers over a range of reduced hydrations and sugar concentrations. Stacked lipid bilayers at reduced hydration provide a suitable system to mimic environmental dehydration effects, as well as a suitable system for direct probing of sugar locations by neutron membrane diffraction. Sugar distributions show that sucrose and trehalose both behave as typical uncharged solutes, largely excluded from the lipid bilayers regardless of sugar identity, and with no correlation between sugar distribution and the lipid headgroup position as the hydration is changed. These results are discussed in terms of current opinions about cryo- and anhydro-protection mechanisms.

Neutron scattering from myelin revisited: bilayer asymmetry and water-exchange kinetics.

Rapid nerve conduction in the central and peripheral nervous systems (CNS and PNS, respectively) of higher vertebrates is brought about by the ensheathment of axons with myelin, a lipid-rich, multilamellar assembly of membranes. The ability of myelin to electrically insulate depends on the regular stacking of these plasma membranes and on the presence of a number of specialized membrane-protein assemblies in the sheath, including the radial component, Schmidt-Lanterman incisures and the axo-glial junctions of the paranodal loops. The disruption of this fine-structure is the basis for many demyelinating neuropathies in the CNS and PNS. Understanding the processes that govern myelin biogenesis, maintenance and destabilization requires knowledge of myelin structure; however, the tight packing of internodal myelin and the complexity of its junctional specializations make myelin a challenging target for comprehensive structural analysis. This paper describes an examination of myelin from the CNS and PNS using neutron diffraction. This investigation revealed the dimensions of the bilayers and aqueous spaces of myelin, asymmetry between the cytoplasmic and extracellular leaflets of the membrane, and the distribution of water and exchangeable hydrogen in internodal multilamellar myelin. It also uncovered differences between CNS and PNS myelin in their water-exchange kinetics.

Small-angle neutron scattering studies of hemoglobin confined inside silica tubes of varying sizes.

In addition to the chemical nature of the surface, the dimensions of the confining host exert a significant influence on confined protein structures; this results in immense biological implications, especially those concerning the enzymatic activities of the protein. This study probes the structure of hemoglobin (Hb), a model protein, confined inside silica tubes with pore diameters that vary by one order of magnitude (≈20-200 nm). The effect of confinement on the protein structure is probed by comparison with the structure of the protein in solution. Small-angle neutron scattering (SANS), which provides information on protein tertiary and quaternary structures, is employed to study the influence of the tube pore diameter on the structure and configuration of the confined protein in detail. Confinement significantly influences the structural stability of Hb and the structure depends on the Si-tube pore diameter. The high radius of gyration (Rg) and polydispersity of Hb in the 20 nm diameter Si-tube indicates that Hb undergoes a significant amount of aggregation. However, for Si-tube diameters greater or equal to 100 nm, the Rg of Hb is found to be in very close proximity to that obtained from the protein data bank (PDB) reported structure (Rg of native Hb=23.8 Å). This strongly indicates that the protein has a preference for the more native-like non-aggregated state if confined inside tubes of diameter greater or equal to 100 nm. Further insight into the Hb structure is obtained from the distance distribution function, p(r), and ab initio models calculated from the SANS patterns. These also suggest that the Si-tube size is a key parameter for protein stability and structure.

Biochemical and Phylogenetic Analysis of Italian Phaseolus vulgaris Cultivars as Sources of α-Amylase and α-Glucosidase Inhibitors.

Phaseolus vulgaris α-amylase inhibitor (α-AI) is a protein that has recently gained commercial interest, as it inhibits mammalian α-amylase activity, reducing the absorption of dietary carbohydrates. Numerous studies have reported the efficacy of preparations based on this protein on the control of glycaemic peaks in type-2 diabetes patients and in overweight subjects. A positive influence on microbiota regulation has also been described. In this work, ten insufficiently studied Italian P. vulgaris cultivars were screened for α-amylase- and α-glucosidase-inhibiting activity, as well as for the absence of antinutritional compounds, such as phytohemagglutinin (PHA). All the cultivars presented α-glucosidase-inhibitor activity, while α-AI was missing in two of them. Only the Nieddone cultivar (ACC177) had no haemagglutination activity. In addition, the partial nucleotide sequence of the α-AI gene was identified with the degenerate hybrid oligonucleotide primer (CODEHOP) strategy to identify genetic variability, possibly linked to functional α-AI differences, expression of the α-AI gene, and phylogenetic relationships. Molecular studies showed that α-AI was expressed in all the cultivars, and a close similarity between the Pisu Grogu and Fasolu cultivars' α-AI and α-AI-4 isoform emerged from the comparison of the partially reconstructed primary structures. Moreover, mechanistic models revealed the interaction network that connects α-AI with the α-amylase enzyme characterized by two interaction hotspots (Asp38 and Tyr186), providing some insights for the analysis of the α-AI primary structure from the different cultivars, particularly regarding the structure-activity relationship. This study can broaden the knowledge about this class of proteins, fuelling the valorisation of Italian agronomic biodiversity through the development of commercial preparations from legume cultivars.

Experimental-theoretical study of laccase as a detoxifier of aflatoxins.

We investigate laccase-mediated detoxification of aflatoxins, fungal carcinogenic food contaminants. Our experimental comparison between two aflatoxins with similar structures (AFB1 and AFG2) shows significant differences in laccase-mediated detoxification. A multi-scale modeling approach (Docking, Molecular Dynamics, and Density Functional Theory) identifies the highly substrate-specific changes required to improve laccase detoxifying performance. We employ a large-scale density functional theory-based approach, involving more than 7000 atoms, to identify the amino acid residues that determine the affinity of laccase for aflatoxins. From this study we conclude: (1) AFB1 is more challenging to degrade, to the point of complete degradation stalling; (2) AFG2 is easier to degrade by laccase due to its lack of side products and favorable binding dynamics; and (3) ample opportunities to optimize laccase for aflatoxin degradation exist, especially via mutations leading to π-π stacking. This study identifies a way to optimize laccase for aflatoxin bioremediation and, more generally, contributes to the research efforts aimed at rational enzyme optimization.

Assembling wormlike micelles in tubular nanopores by tuning surfactant-wall interactions.

Threadlike molecular assemblies are excluded from narrow pores unless attractive interactions with the confining pore walls compensate for the loss of configurational entropy. Here we show that wormlike surfactant micelles can be assembled in the 8 nm tubular nanopores of SBA-15 silica by adjusting the surfactant-pore-wall interactions. The modulation of the interactions was achieved by coadsorption of a surface modifier that also provides control over the partitioning of wormlike aggregates between the bulk solution and the pore space. We anticipate that the concept of tuning the interactions with the pore wall will be applicable to a wide variety of self-assembling molecules and pores.

Impact of lyoprotectors on protein-protein separation in the solid state: Neutron- and X-ray-scattering investigation.

BACKGROUND: Polyhydroxycompounds (PHC) are used as lyoprotectors to minimize aggregation of pharmaceutical proteins during freeze-drying and storage. METHODS: Lysozyme/PHC mixtures with 1:1 and 1:3 (w/w) ratios are freeze-dried from either H2O or D2O solutions. Disaccharides (sucrose and trehalose), monosaccharide (glucose), and sugar alcohol (sorbitol) are used in the study. Small-angle neutron and X-ray scattering (SANS and SAXS) are applied to study protein-protein interaction in the freeze-dried samples. RESULTS: Protein interaction peak in the freeze-dried mixtures has been detected by both SANS (D2O-based samples only) and SAXS (both D2O- and H2O-based). In the 1:1 mixtures, protein separation distances are similar (center-of-mass distance of approx. 31 Å) between all lyoprotectors studied. Mixtures with a higher content of the disaccharides (1:3 ratio) have a higher separation distance of approx 40 Å. The higher separation could reduce protein-protein contacts and therefore be associated with less favourable aggregation conditions. In the 1:3 mixtures with glucose and sorbitol, complex SANS and SAXS/WAXS patterns are observed. The pattern for the glucose sample indicate two populations of lysozyme molecules, while the origin of multiple SAXS peaks in the lysozyme/sorbitol 1:3 mixture is uncertain. CONCLUSIONS: Protein-protein separation distance is determined predominantly by the lyoprotector/protein weight ratio. GENERAL SIGNIFICANCE: Use of SANS and SAXS improves understanding of mechanisms of protein stabilization by sugars in freeze-dried formulations, and provide a tool to verify hypothesis on relationship between protein/protein separation and aggregation propensity in the dried state.

Probing the mutational landscape of the SARS-CoV-2 spike protein via quantum mechanical modeling of crystallographic structures.

We employ a recently developed complexity-reduction quantum mechanical (QM-CR) approach, based on complexity reduction of density functional theory calculations, to characterize the interactions of the SARS-CoV-2 spike receptor binding domain (RBD) with ACE2 host receptors and antibodies. QM-CR operates via ab initio identification of individual amino acid residue's contributions to chemical binding and leads to the identification of the impact of point mutations. Here, we especially focus on the E484K mutation of the viral spike protein. We find that spike residue 484 hinders the spike's binding to the human ACE2 receptor (hACE2). In contrast, the same residue is beneficial in binding to the bat receptor Rhinolophus macrotis ACE2 (macACE2). In agreement with empirical evidence, QM-CR shows that the E484K mutation allows the spike to evade categories of neutralizing antibodies like C121 and C144. The simulation also shows how the Delta variant spike binds more strongly to hACE2 compared to the original Wuhan strain, and predicts that a E484K mutation can further improve its binding. Broad agreement between the QM-CR predictions and experimental evidence supports the notion that ab initio modeling has now reached the maturity required to handle large intermolecular interactions central to biological processes.