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SARS-CoV-2 and HCoV-OC43 belong to the same β genus of the Coronaviridae family. SARS-CoV-2 was responsible for the recent COVID-19 pandemic, and HCoV-OC43 is the etiological agent of mild upper respiratory tract infections. SARS-COV-2 and HCoV-OC43 co-infections were found in children with respiratory symptoms during the COVID-19 pandemic. The two β-coronaviruses share a high degree of homology between the 3CLpro active sites, so much so that the safer HCoV-OC43 has been suggested as a tool for the identification of new anti-SARS-COV-2 agents. Compounds 5 and 24 inhibited effectively both Wuhan and British SARS-CoV-2 patient isolates in Vero E6 cells and the HCoV-OC43 in MRC-5 cells at low micromolar concentrations. The inhibition was apparently exerted via targeting the 3CLpro active sites of both viruses. Compounds 5 and 24 at 100 μM inhibited the SARS-CoV-2 3CLpro activity of 61.78 and 67.30%, respectively. These findings highlight 5 and 24 as lead compounds of a novel class of antiviral agents with the potential to treat SARS-COV-2 and HCoV-OC43 infections.

After harvesting of cones used for beer production, the remaining hop vegetative biomass requires disposal. The hop plant contains bioactive compounds in all its parts-cones, leaves, and roots-exhibiting interesting antioxidant, antiviral, and antibacterial properties. In this work, extracts obtained from hop leaves, a plant material often neglected in the hop cultivation, have been investigated; the qualitative UHPLC-MS/MS and GC-TOF-MS characterization revealed the presence of bioactive compounds such as polyphenols, α- and β-acids and terpenes are present. The extract retained antioxidant activity, as verified by Folin-Ciocalteu, DPPH, ABTS and FRAP assays, and demonstrated some antimicrobial activity when combined with antibiotics, particularly against Gram-positive bacterial strains. Additionally, the extracts showed an ability to interact with proteins as human insulin, amyloid beta peptide, mucin and bovine serum albumin (BSA), has been detected, indicating their potential to counteract inflammatory processes and protect against Alzheimer's disease. These findings suggest that hop vegetative biomass, typically considered waste, can be potentially transformed into a valuable resource with applications in various fields, from nutraceuticals to pharmaceuticals and cosmetics, aligning with a circular economy perspective., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Ltd.)

Hypothesis: Renal calculi (kidney stones) are mainly made by calcium oxalate and can cause different complications including malfunction of the kidney. The most important urinary stone inhibitors are citrate molecules. Unfortunately, the amount of citrate reaching the kidney after oral ingestion is low. We hypothesized that nanoparticles of polyallylamine hydrochloride (CIT-PAH) carrying citrate ions could simultaneously deliver citrates while PAH would complex oxalate triggering dissolution and removal of CaOx nanocrystals., Experiments: We successfully prepared nanoparticles of citrate ions with polyallylamine hydrochloride (CIT-PAH), PAH with oxalate (OX-PAH) and characterize them by Small Angle X ray Scattering (SAXS), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and NMR. Dissolution of CaOx nanocrystals in presence of CIT-PAH have been followed with Wide Angle Xray Scattering (WAXS), DLS and Confocal Raman Microscopy. Raman spectroscopy was used to study the dissolution of crystals in synthetic urine samples. The release of citrate from CIT-PAH was followed by diffusion NMR. Molecular dynamics (MD) simulations were carried out to study the interaction of CIT and OX ions with PAH., Findings: CIT-PAH nanoparticles dissolves CaOx nanocrystals as shown by NMR, DLS, TEM and WAXS in water and by Raman spectroscopy in artificial human urine. WAXS and Raman show that the crystal structure of CaOx disappears in the presence of CIT-PAH. DLS shows that the time required for CaOX dissolution will depend on the concentration of CIT-PAH NPs. NMR proves that citrate ions are released from the CIT PAH NPs during CaOX dissolution, MD simulations showed that oxalates exhibit a stronger interaction for PAH than citrate, explaining the removal of oxalate ions and replacement of the citrate in the polymer nanoparticles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The main proteases M pro are a group of highly conserved cysteine hydrolases in β-coronaviruses. They have been demonstrated to play an unavoidable role in viral replication, and consequently they have been suggested as key targets for treating coronavirus-caused infectious diseases, mainly from the COVID-19 epidemic. Since the most functional form for M pro enzymatic activity is associated to its homodimer, compounds inhibiting dimerization should also inhibit catalytic activity. We show how PIR-SEIRA (Plasmonic Internal Reflection-Surface Enhanced InfraRed Absorption) spectroscopy can be a noteworthy technique to study proteins subtle structural variations associated to inhibitor binding. Nanoantennas arrays can selectively confine and enhance electromagnetic field via localized plasmonic resonances, thus promoting ultrasensitive detection of biomolecules in close proximity of nanoantenna arrays and enabling the effective investigation of protein monolayers. By adopting this approach, reflection measurements conducted under back illumination of nanoantennas allow to probe anchored protein monolayers, with minimum contribution of environmental buffer molecules. PIR-SEIRA spectroscopy on M pro was carried out by ad hoc designed devices, resonating in the spectral region of Amide I and Amide II bands. We evaluated here the structure of anchored monomers and dimers in different buffered environment and in presence of a newly designed M pro inhibitor. Experimental results show that dimerization is not associated to relevant backbone rearrangements of the protein at secondary structure level, and even if the compound inhibits the dimerization, it is not effective at breaking preformed dimers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

N -Acetylgalactosamine-6-sulfatase (GALNS) is an enzyme whose deficiency is related to the lysosomal storage disease Morquio A. For the development of effective therapeutic approaches against this disease, the design of suitable enzyme enhancers ( i.e. pharmacological chaperones) is fundamental. The natural substrates of GALNS are the glycosaminoglycans keratan sulfate and chondroitin 6-sulfate, which mainly display repeating units of sulfated carbohydrates. With a biomimetic approach, gold nanoparticles (AuNPs) decorated with simple monosaccharides, sulfated ligands (homoligand AuNPs), or both monosaccharides and sulfated ligands (mixed-ligand AuNPs) were designed here as multivalent inhibitors of GALNS. Among the homoligand AuNPs, the most effective inhibitors of GALNS activity are the β-D-galactoside-coated AuNPs. In the case of mixed-ligand AuNPs, β-D-galactosides/sulfated ligands do not show better inhibition than the β-D-galactoside-coated AuNPs. However, a synergistic effect is observed for α-D-mannosides in a mixed-ligand coating with sulfated ligands that reduced IC 50 by one order of magnitude with respect to the homoligand α-D-mannoside-coated AuNPs. SAXS experiments corroborated the association of GALNS with β-D-galactoside AuNPs. These AuNPs are able to restore the enzyme activity by almost 2-fold after thermal denaturation, indicating a potential chaperoning activity towards GALNS. This information could be exploited for future development of nanomedicines for Morquio A. The recent implications of GALNS in cancer and neuropathic pain make these kinds of multivalent bionanomaterials of great interest towards multiple therapies.

Cyanidin 3-O-glucoside (CND) is a frequently-used anthocyanin that has excellent antioxidant properties but a limited bioavailability in bloodstream. Complexation of CND with alginate can improve its therapeutic outcome. Here we have studied the complexation of CND with alginate under a range of pH values from 2.5 to 5. CND is positively charged at low pH, and becomes neutral, and then negatively charged as pH increases. CND/alginate complexation was studied by dynamic light scattering, transmission electron microscopy, small angle X-ray scattering, STEM, UV-Vis spectroscopy and circular dichroism (CD). CND/alginate complexes at pH 4.0 and 5.0 form chiral fibres with a fractal structure. At these pH values, CD spectra show very intense bands, which are inverted compared with free CND. Complexation at lower pH results in disordered polymer structures and CD spectra show the same features as for CND in solution. Molecular dynamics simulations suggest the formation of parallel CND dimers through complexation with alginate at pH 3.0, while at pH 4.0 CND dimers form in a cross like arrangement., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Hypothesis: Modification of polyallylamine hydrochloride (PAH) with heterobifunctional low molecular weight polyethylene glycol (PEG) (600 and 1395 Da), and subsequent attachment of mannose, glucose, or lactose sugars to PEG, can lead to formation of polyamine phosphate nanoparticles (PANs) with lectin binding affinity and narrow size distribution., Experiments: Size, polydispersity, and internal structure of glycosylated PEGylated PANs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). Fluorescence correlation spectroscopy (FCS) was used to study the association of labelled glycol-PEGylated PANs. The number of polymer chains forming the nanoparticles was determined from the changes in amplitude of the cross-correlation function of the polymers after formation of the nanoparticles. SAXS and fluorescence cross-correlation spectroscopy were used to investigate the interaction of PANs with lectins: concanavalin A with mannose modified PANs, and jacalin with lactose modified ones., Findings: Glyco-PEGylated PANs are highly monodispersed, with diameters of a few tens of nanometers and low charge, and a structure corresponding to spheres with Gaussian chains. FCS shows that the PANs are single chain nanoparticles or formed by two polymer chains. Concanavalin A and jacalin show specific interactions for the glyco-PEGylated PANs with higher affinity than bovine serum albumin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The study of the interaction between lipid membranes and amyloidogenic peptides is a turning point for understanding the processes involving the cytotoxicity of peptides involved in neurodegenerative diseases. In this work, we perform an experimental study of model membrane-lysozyme interaction to understand how the formation of amyloid fibrils can be affected by the presence of polar and zwitterionic phospholipid molecules (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine [POPC] and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol [POPG]). The study was conducted above and below the critical micellar concentration (CMC) using dynamic light scattering (DLS), atomic force microscopy (AFM), UV-Vis spectrophotometry, and the quartz crystal microbalance (QCM). Our results show that the presence of phospholipids appears to be a factor favoring the formation of amyloid aggregates. Spectrophotometric and DLS data revealed that the quantity of β -structure increases in the presence of POPG and POPC at different concentrations. The presence of POPG and POPC increases the speed of the nucleation process, without altering the overall structures of the fibrillar final products., (© 2023 the author(s), published by De Gruyter.)

Diatoms represent the most abundant and diversified class of primary producers in present oceans; their distinctive trait is the ability to incorporate silicic acid in a silica outer shell called frustule. Numerous adaptative functions are ascribed to frustules, including the control of vertical movements through the water column; this indirectly determines cell access to fundamental resources such as light and nutrients, and favors diatom escape from predators. At the same time, light guides phototroph movements in the water column by affecting cell density (e.g., by modulating Si deposition in diatoms, vacuole volume, and/or solution). We investigated how the tremendous diversity in morphology and silicification that characterizes the frustule and the crucial role of light in diatom spatial distribution govern diatom sinking capacity. To test their integrative effects, we acclimated four diatoms distinguished by frustule traits ( Chaetoceros muelleri , Conticribra weissflogii , Phaeodactylum tricornutum , and Cylindrotheca fusiformis ) to different light conditions and evaluated their physiological performance in terms of growth, elemental composition, morphological changes, and their in vivo sinking capacity. What emerged from this study was that silicification, more than other morphological characteristics, controls species vertical movements, while a higher energy availability enhances cell floating independently from the silica content., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Petrucciani, Moretti, Ortore and Norici.)

The use of liposomes as drug delivery systems emerged in the last decades in view of their capacity and versatility to deliver a variety of therapeutic agents. By means of small-angle neutron scattering (SANS), we performed a detailed characterization of liposomes containing outer membrane protein F (OprF), the main porin of the Pseudomonas aeruginosa bacterium outer membrane. These OprF-liposomes are the basis of a novel vaccine against this antibiotic-resistant bacterium, which is one of the main hospital-acquired pathogens and causes each year a significant number of deaths. SANS data were analyzed by a specific model we created to quantify the crucial information about the structure of the liposome containing OprF, including the lipid bilayer structure, the amount of protein in the lipid bilayer, the average protein localization, and the effect of the protein incorporation on the lipid bilayer. Quantification of such structural information is important to enhance the design of liposomal delivery systems for therapeutic applications.

The degradation of mesoporous silica nanoparticles (MSNs) in the biological milieu due to silica hydrolysis plays a fundamental role for the delivery of encapsulated drugs and therapeutics. However, little is known on the evolution of the pore arrangement in the MSNs in biologically relevant conditions. Small Angle X-ray scattering (SAXS) studies were performed on unmodified and PEGylated MSNs with a MCM-48 pore structure and average sizes of 140 nm, exposed to simulated body fluid solution (SBF) at pH 7.4 for different time intervals from 30 min to 24 h. Experiments were performed with silica concentrations below, at and over 0.14 mg/mL, the saturation concentration of silica in water at physiological temperature. At silica concentrations of 1 mg/mL (oversaturation), unmodified MSNs show variation in interpore distances over 6 h exposure to SBF, remaining constant thereafter. A decrease in radius of gyration is observed over the same time. Mesoporosity and radius of gyration of unmodified MSNs remain then unchanged up to 24 h. PEGylated MSNs at 1 mg/mL concentration show a broader diffraction peak but no change in the position of the peak is observed following 24 h exposure to SBF. PEGylated MSNs at 0.01 mg/mL show no diffraction peaks already after 30 min exposure to SBF, while at 0.14 mg/mL a small diffraction peak is present after 30 min exposure but disappears after 1 h., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The cblC disease is an inborn disorder of the vitamin B12 (cobalamin, Cbl) metabolism characterized by methylmalonic aciduria and homocystinuria. The clinical consequences of this disease are devastating and, even when early treated with current therapies, the affected children manifest symptoms involving vision, growth, and learning. The illness is caused by mutations in the gene codifying for MMACHC, a 282aa protein that transports and transforms the different Cbl forms. Here we present data on the structural properties of the truncated protein p.R132X resulting from the c.394C > T mutation that, along with c.271dupA and c.331C > T, is among the most common mutations in cblC. Although missing part of the Cbl binding domain, p.R132X is associated to late-onset symptoms and, therefore, it is supposed to retain residual function. However, to our knowledge structural-functional studies on c.394C > T mutant aimed at verifying this hypothesis are still lacking. By using a biophysical approach including Circular Dichroism, fluorescence, Small Angle X-ray Scattering, and Molecular Dynamics, we show that the mutant protein MMACHC-R132X retains secondary structure elements and remains compact in solution, partly preserving its binding affinity for Cbl. Insights on the fragile stability of MMACHC-R132X-Cbl are provided., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Taurine is an important organic osmolyte in mammalian cells, and it weakens inflammation and oxidative stress mediated injuries in some diseases. Recently, taurine has been demonstrated to play a therapeutic role against neurodegenerative disorders, although its parallel involvement in several biochemical mechanisms makes not clear taurine specific role in these diseases. Furthermore, the stabilizing effect of this molecule in terms of protein stability is known, but not deeply investigated. In this work we explore by Circular Dichroism the stabilizing impact of taurine in lysozyme thermal denaturation and its influence in lysozyme aggregation into amyloid fibrils. Taurine even at low concentration modifies protein-protein interactions in lysozyme native state, as revealed by Small Angle X-ray Scattering experiments, and alters the amyloid aggregation pattern without completely inhibiting it, as confirmed by UV/Vis spectroscopy with Congo Red and by Atomic Force Microscopy. Evaluation of the cytotoxicities of the amyloid fibrils grown in presence or in absence of taurine is investigated on SH-SY5Y neuroblastoma cells., Competing Interests: The authors declare no conflicts of interest.

Many proteins are usually not stable under different stresses, such as temperature and pH variations, mechanical stresses, high concentrations, and high saline contents, and their transport is always difficult, because they need to be maintained in a cold regime, which is costly and very challenging to achieve in remote areas of the world. For this reason, it is extremely important to find stabilizing agents that are able to preserve and protect proteins against denaturation. In the present work, we investigate, by extensively using synchrotron small-angle X-ray scattering experiments, the stabilization effect of five different sugar-derived compounds developed at ExtremoChem on two model proteins: myoglobin and insulin. The data analysis, based on a novel method that combines structural and thermodynamic features, has provided details about the physical-chemical processes that regulate the stability of these proteins in the presence of stabilizing compounds. The results clearly show that some modified sugars exert a greater stabilizing effect than others, being able to maintain the active forms of proteins at temperatures higher than those in which proteins, in the absence of stabilizers, reach denatured states.

Background: The phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. It is formed by a protein cascade in which the first two proteins are general (namely enzyme I, EI, and the histidine phosphocarrier protein, HPr) and the others are sugar-specific permeases; the active site of HPr is His15. The HPr kinase/phosphorylase (HPrK/P), involved in the use of carbon sources in Gram-positive, phopshorylates HPr at a serine. The regulator of sigma D protein (Rsd) also binds to HPr. We are designing specific fragments of HPr, which can be used to interfere with those protein-protein interactions (PPIs), where the intact HPr intervenes., Methods: We obtained a fragment (HPr48) comprising the first forty-eight residues of HPr. HPr48 was disordered as shown by fluorescence, far-ultraviolet (UV) circular dichroism (CD), small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR)., Results: Secondary structure propensities, from the assigned backbone nuclei, further support the unfolded nature of the fragment. However, HPr48 was capable of binding to: (i) the N-terminal region of EI, EIN; (ii) the intact Rsd; and, (iii) HPrK/P, as shown by fluorescence, far-UV CD, NMR and biolayer interferometry (BLI). The association constants for each protein, as measured by fluorescence and BLI, were in the order of the low micromolar range, similar to those measured between the intact HPr and each of the other macromolecules., Conclusions: Although HPr48 is forty-eight-residue long, it assisted antibiotics to exert antimicrobial activity., General Significance: HPr48 could be used as a lead compound in the development of new antibiotics, or, alternatively, to improve the efficiency of existing ones., (Copyright © 2021 Elsevier B.V. All rights reserved.)

An approach for reducing toxicity and enhancing therapeutic potential of supramolecular polyamine phosphate nanoparticles (PANs) through PEGylation of polyamines before their assembly into nanoparticles is presented here. It is shown that the number of polyethylene glycol (PEG) chains for polyamine largely influence physico-chemical properties of PANs and their biological endpoints. Poly(allylamine hydrochloride) (PAH) are functionalized through carbodiimide chemistry with three ratios of PEG molecules per PAH chain: 0.1, 1, and 10. PEGylated PAH is then assembled into PANs by exposing the polymer to phosphate buffer solution. PANs decrease size and surface charge with increasing PEG ratios as evidenced by dynamic light scattering and zeta potential measurements, with the ten PEG/PAH ratio PANs having practically zero charge. Small angle X-ray scattering (SAXS) proves that PEG chains form a shell around a polyamine core, which is responsible for the screening of positive charges. MTT experiments show that the screening of amine groups decreases nanoparticle toxicity, with the lowest toxicity for the 10 PEG/PAH ratio. Fluorescence correlation spectroscopy (FCS) proves less interaction with proteins for PEGylated PANs. Positron emission tomography (PET) imaging of 18 F labelled PANs shows longer circulation time in healthy mice for PEGylated PANs than non-PEGylated ones., (© 2021 Wiley-VCH GmbH.)

The maturation of coronavirus SARS-CoV-2, which is the etiological agent at the origin of the COVID-19 pandemic, requires a main protease M pro to cleave the virus-encoded polyproteins. Despite a wealth of experimental information already available, there is wide disagreement about the M pro monomer-dimer equilibrium dissociation constant. Since the functional unit of M pro is a homodimer, the detailed knowledge of the thermodynamics of this equilibrium is a key piece of information for possible therapeutic intervention, with small molecules interfering with dimerization being potential broad-spectrum antiviral drug leads. In the present study, we exploit Small Angle X-ray Scattering (SAXS) to investigate the structural features of SARS-CoV-2 M pro in solution as a function of protein concentration and temperature. A detailed thermodynamic picture of the monomer-dimer equilibrium is derived, together with the temperature-dependent value of the dissociation constant. SAXS is also used to study how the M pro dissociation process is affected by small inhibitors selected by virtual screening. We find that these inhibitors affect dimerization and enzymatic activity to a different extent and sometimes in an opposite way, likely due to the different molecular mechanisms underlying the two processes. The M pro residues that emerge as key to optimize both dissociation and enzymatic activity inhibition are discussed.

The histidine phosphocarrier protein (HPr) kinase/phosphorylase (HPrK/P) modulates the phosphorylation state of the HPr protein, and it is involved in the use of carbon sources by Gram-positive bacteria. Its X-ray structure, as concluded from crystals of proteins from several species, is a hexamer; however, there are no studies about its conformational stability, and how its structure is modified by the pH. We have embarked on the conformational characterization of HPrK/P of Bacillus subtilis (bsHPrK/P) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, small-angle X-ray-scattering (SAXS) and dynamic light-scattering (DLS)). bsHPrK/P was mainly a hexamer in solution at pH 7.0, in the presence of phosphate. The protein had a high conformational stability, with an apparent thermal denaturation midpoint of ~70 °C, at pH 7.0, as monitored by fluorescence and CD. The protein was very pH-sensitive, precipitated between pH 3.5 and 6.5; below pH 3.5, it had a molten-globule-like conformation; and it acquired a native-like structure in a narrow pH range (between pH 7.0 and 8.0). Guanidinium hydrochloride (GdmCl) denaturation occurred through an oligomeric intermediate. On the other hand, urea denaturation occurred as a single transition, in the range of concentrations between 1.8 and 18 µM, as detected by far-UV CD and fluorescence.

The in solution synchrotron small-angle X-ray scattering SAXS technique has been used to investigate an intrinsically disordered protein (IDP) related to Parkinson's disease, the α-synuclein (α-syn), in prefibrillar diluted conditions. SAXS experiments have been performed as a function of temperature and concentration on the wild type (WT) and on the three pathogenic mutants G51D, E46K, and A53T. To identify the conformers that populate WT α-syn and the pathogenic mutants in prefibrillar conditions, scattering data have been analyzed by a new variational bayesian weighting method (VBWSAS) based on an ensemble of conformers, which includes unfolded monomers, trimers, and tetramers, both in helical-rich and strand-rich forms. The developed VBWSAS method uses a thermodynamic scheme to account for temperature and concentration effects and considers long-range protein-protein interactions in the framework of the random phase approximation. The global analysis of the whole set of data indicates that WT α-syn is mostly present as unfolded monomers and trimers (helical-rich trimers at low T and strand-rich trimers at high T ), but not tetramers, as previously derived by several studies. On the contrary, different conformer combinations characterize mutants. In the α-syn G51D mutant, the most abundant aggregates at all the temperatures are strand-rich tetramers. Strand-rich tetramers are also the predominant forms in the A53T mutant, but their weight decreases with temperature. Only monomeric conformers, with a preference for the ones with the smallest sizes, are present in the E46K mutant. The derived conformational behavior then suggests a different availability of species prone to aggregate, depending on mutation, temperature, and concentration and accounting for the different neurotoxicity of α-syn variants. Indeed, this approach may be of pivotal importance to describe conformational and aggregational properties of other IDPs.

We evaluate, by means of synchrotron small-angle X-ray scattering, the shape and mutual interactions of DNA tetravalent nanostars as a function of temperature in both the gas-like state and across the gel transition. To this end, we calculate the form factor from coarse-grained molecular dynamics simulations with a novel method that includes hydration effects; we approximate the radial interaction of DNA nanostars as a hard-sphere potential complemented by a repulsive and an attractive Yukawa term; and we predict the structure factors by exploiting the perturbative random phase approximation of the Percus-Yevick equation. Our approach enables us to fit all the data by selecting the particle radius and the width and amplitude of the attractive potential as free parameters. We determine the evolution of the structure factor across gelation and detect subtle changes of the effective interparticle interactions, that we associate to the temperature and concentration dependence of the particle size. Despite the approximations, the approach here adopted offers new detailed insights into the structure and interparticle interactions of this fascinating system.

Protein aggregation into amyloid fibrils is a phenomenon that attracts attention from a wide and composite part of the scientific community. Indeed, the presence of mature fibrils is associated with several neurodegenerative diseases, and in addition these supramolecular aggregates are considered promising self-assembling nanomaterials. In this framework, investigation on the effect of cosolutes on protein propensity to aggregate into fibrils is receiving growing interest, and new insights on this aspect might represent valuable steps towards comprehension of highly complex biological processes. In this work we studied the influence exerted by the osmolyte trehalose on fibrillation of two model proteins, that is, lysozyme and insulin, investigated during concomitant variation of the solution ionic strength due to NaCl. In order to monitor both secondary structures and the overall tridimensional conformations, we have performed UV spectroscopy measurements with Congo Red, Circular Dichroism, and synchrotron Small Angle X-ray Scattering. For both proteins we describe the effect of trehalose in changing the fibrillation pattern and, as main result, we observe that ionic strength in solution is a key factor in determining trehalose efficiency in slowing down or blocking protein fibrillation. Ionic strength reveals to be a competitive element with respect to trehalose, being able to counteract its inhibiting effects toward amyloidogenesis. Reported data highlight the importance of combining studies carried out on cosolutes with valuation of other physiological parameters that may affect the aggregation process. Also, the obtained experimental results allow to hypothesize a plausible mechanism adopted by the osmolyte to preserve protein surface and prevent protein fibrillation.

Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS is regulated, among other mechanisms, by protein-protein interactions with a 65-residue-long, intrinsically disordered protein (IDP), named IF7. IDPs explore diverse conformations in their free states and, in some cases, in their molecular complexes. We used both nuclear magnetic resonance (NMR) at 11.7 T and small angle X-ray scattering (SAXS) to study the size and the dynamics in the picoseconds-to-nanosecond (ps-ns) timescale of: (i) isolated IF7; and (ii) the IF7/GS complex. Our SAXS findings, together with MD results, show: (i) some of the possible IF7 structures in solution; and, (ii) that the presence of IF7 affected the structure of GS in solution. The joint use of SAXS and NMR shows that movements of each amino acid of IF7 were uncorrelated with those of its neighbors. Residues of IF7 with the largest values of the relaxation rates (R 1 , R 2 and η xy ), in the free and bound species, were mainly clustered around: (i) the C terminus of the protein; and (ii) Ala30. These residues, together with Arg8 (which is a hot-spot residue in the interaction with GS), had a restricted mobility in the presence of GS. The C-terminal region, which appeared more compact in our MD simulations of isolated IF7, seemed to be involved in non-native contacts with GS that help in the binding between the two macromolecules., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The hierarchical process of guanosine (G) self-assembly, leading in aqueous solution and in the presence of metal cations to the formation of G-quadruplexes, represents an intriguing topic both for the biological correlation with telomerase activity and for the nano-technological applications, as demonstrated by the current measured in a quadruplex wire 100 nm long. Similar to G-rich DNA sequences and G-oligonucleotides, the guanosine 5'-monophosphate (GMP) self-aggregates in water to form quadruplexes. However, due to the absence of a covalent axial backbone, this system can be very useful to understand the chemical-physical conditions that govern the guanosine supramolecular aggregation. We have then investigated by in-solution Synchrotron Small Angle X-ray Scattering technique the role of different cations in promoting the quadruplex formation as a function of concentration and temperature. Results show how potassium, with its peculiar biological traits, favours the G-quadruplex elongation process in respect to other cations (Na + , but also NH 4 + and Li + ), determining the longest particles in solution. Moreover, the formation and the elongation of G-quadruplexes have been demonstrated to be controlled by both GMP concentration and excess cation content, even if they specifically contribute to these processes in different ways. The occurrence of condensed liquid crystalline phases was also detected, proving that excess cations play also unspecific effects on the effective charges on the G-quadruplex surface.

Dynamic Light Scattering (DLS), Small Angle X-ray Scattering (SAXS) and Transmission Electron Microscopy (TEM) are physical techniques widely employed to characterize the morphology and the structure of vesicles such as liposomes or human extracellular vesicles (exosomes). Bacterial extracellular vesicles are similar in size to human exosomes, although their function and membrane properties have not been elucidated in such detail as in the case of exosomes. Here, we applied the above cited techniques, in synergy with the thermotropic characterization of the vesicles lipid membrane using a turbidimetric technique to the study of vesicles produced by Gram-negative bacteria (Outer Membrane Vesicles, OMVs) grown at different temperatures. This study demonstrated that our combined approach is useful to discriminate vesicles of different origin or coming from bacteria cultured under different experimental conditions. We envisage that in a near future the techniques employed in our work will be further implemented to discriminate complex mixtures of bacterial vesicles, thus showing great promises for biomedical or diagnostic applications., (Copyright © 2020 Sarra, Celluzzi, Bruno, Ricci, Sennato, Ortore, Casciardi, Del Chierico, Postorino, Bordi and Masotti.)

Lipophilic guanines (LipoGs) in aprotic solvents undergo different self-assembly processes based on different H-bonded motifs. Cylindrical nanotubes made by π-π stacked guanine tetramers (G-quadruplexes) and flat, tape-like aggregates (G-ribbons) have been observed depending on the presence of alkali metal ions. To obtain information on the structural properties and stability of these LipoG aggregates, Small-Angle X-ray Scattering (SAXS) experiments have been performed in dodecane, both in the presence and in the absence of potassium ions. As a result, the occurrence of the two different metallo-responsive architectures (nanoribbons or columnar nanotubes) was confirmed and we reported here for the first time a systematic study on the dependence of the aggregate properties on composition, temperature and molecular unit structure. Even if dodecane was selected to favour LipoG solubility, a strong tendency to self-organize into ordered lyotropic phases was indeed detected.

Silencing RNA (siRNA) technologies attract significant interest as a therapeutic tool for a large number of diseases. However, the medical translation of this technology is hampered by the lack of effective delivery vehicles for siRNAs in cytosol that prevent their degradation in the bloodstream. The use of molecular complexes based on polyamines have great potential for siRNA delivery as polyamines can protect the siRNA during circulation and at the same time favor siRNA translocation in cytosol. Here, nanoparticles are prepared by complexation of poly(allylamine hydrochloride) (PAH) and siRNA varying the ratio of nitrogen groups from PAH to phosphate groups from siRNA (N/P ratio). Nanoparticles are characterized by transmission electron microscopy and dynamic light scattering. The stability of complexes of green rhodamine labelled PAH (G-PAH) and Cy5 labelled siRNA (R-siRNA) at different pHs and in cell media is studied by fluorescence cross-correlation spectroscopy (FCCS). FCCS studies show that the nanoparticles are stable at physiological pH and in cell media but they disassemble at acidic pH. An optimal N/P ratio of 2 is identified in terms of stability in media, degradation at endosomal pH and toxicity. The intracellular fate of the complexes is studied following uptake in A549 cells. The cross-correlation between G-PAH and R-siRNA decreases substantially 24 h after uptake, while diffusion times of siRNA decrease indicating that the complexes disassemble, liberating the siRNAs. The release of siRNAs into the cytosol is confirmed with parallel confocal laser scanning microscopy. Flow cytometry studies show that PAH/siRNA nanoparticles are effective at silencing green fluorescent protein expression at low N/P ratios at which polyethylenimine/siRNA shows no significant silencing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 μM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R 1 , R 1ρ , R 2 and η xy ), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Alzheimer's disease is a chronic neurodegenerative disease characterized by the accumulation of pathological aggregates of amyloid beta peptide. Many efforts have been focused on understanding peptide aggregation pathways and on identification of molecules able to inhibit aggregation in order to find an effective therapy. As a result, interest in neuroprotective proteins, such as molecular chaperones, has increased as their normal function is to assist in protein folding or to facilitate the disaggregation and/or clearance of abnormal aggregate proteins. Using biophysical techniques, we evaluated the effects of two chaperones, human Hsp60 and bacterial GroEL, on the fibrillogenesis of Aβ 1-42. Both chaperonins interfere with Aβ 1-42 aggregation, but the effect of Hsp60 is more significant and correlates with its more pronounced flexibility and stronger interaction with ANS, an indicator of hydrophobic regions. Dose-dependent ThT fluorescence kinetics and SAXS experiments reveal that Hsp60 does not change the nature of the molecular processes stochastically leading to the formation of seeds, but strongly delays them by recognition of hydrophobic sites of some peptide species crucial for triggering amyloid formation. Hsp60 reduces the initial chaotic heterogeneity of Aβ 1-42 sample at high concentration regimes. The understanding of chaperone action in counteracting pathological aggregation could be a starting point for potential new therapeutic strategies against neurodegenerative diseases.

Polyamine Phosphate Nanoparticles (PANs) have great potential for the delivery of large therapeutics, such as plasmids and/or siRNAs. The formation of PANs by complexation of Poly(allylamine hydrochloride) (PAH) and phosphate ions from Phosphate Buffer (PB) was studied here, and how it is affected by the presence of phosphate ions from PB and ionic strength. From Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) the critical PB concentration for PANs formation was determined. Below this critical point, Small Angle X-ray Scattering (SAXS) studies revealed that small PAH-phosphate aggregates coexist with not complexed or weakly complexed polymer chains in solution and that the presence of the phosphate ions increases the Kuhn length of the polymer chains until that only spherical aggregates are present in solution. TEM, DLS and SAXS showed the increase of PANs size with ionic strength up to 250 mM NaCl. At higher NaCl concentrations, PANs disassemble into smaller aggregates. Isothermal Titration Calorimetry (ITC) showed that PAN formation is an exothermic process and the association of phosphates below the critical PB concentration is entropically controlled., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The understanding of amyloid β-peptide (Aβ) interactions with cellular membranes is a crucial molecular challenge against Alzheimer's disease. Indeed, Aβ prefibrillar oligomeric intermediates are believed to be the most toxic species, able to induce cellular damages directly by membrane damage. We present a neutron-scattering study on the interaction of large unilamellar vesicles (LUV), as cell membrane models, with both freshly dissolved Aβ and early toxic prefibrillar oligomers, intermediate states in the amyloid pathway. In addition, we explore the effect of coincubating the Aβ-peptide with the chaperonin Hsp60, which is known to strongly interact with it in its aggregation pattern. In fact, the interaction of the LUV with coincubated Aβ/Hsp60, right after mixing and after following the aggregation protocol leading to the toxic intermediates in the absence of Hsp60, is studied. Neutron spin echo experiments show that the interaction with both freshly dissolved and aggregate Aβ species brings about an increase in membrane stiffness, whereas the presence of even very low amounts of Hsp60 (ratio Aβ/Hsp60 = 25:1) maintains unaltered the elastic properties of the membrane bilayer. A coherent interpretation of these results, related to previous literature, can be based on the ability of the chaperonin to interfere with Aβ aggregation, by the specific recognition of the Aβ-reactive transient species. In this framework, our results strongly suggest that early in a freshly dissolved Aβ solution are present some species able to modify the bilayer dynamics, and the chaperonin plays the role of an assistant in such stochastic "misfolding events", avoiding the insult on the membrane as well as the onset of the aggregation cascade.

Bone repair/regeneration is usually investigated through X-ray computed microtomography (μCT) supported by histology of extracted samples, to analyse biomaterial structure and new bone formation processes. Magnetic resonance imaging (μMRI) shows a richer tissue contrast than μCT, despite at lower resolution, and could be combined with μCT in the perspective of conducting non-destructive 3D investigations of bone. A pipeline designed to combine μMRI and μCT images of bone samples is here described and applied on samples of extracted human jawbone core following bone graft. We optimized the coregistration procedure between μCT and μMRI images to avoid bias due to the different resolutions and contrasts. Furthermore, we used an Adaptive Multivariate Clustering, grouping homologous voxels in the coregistered images, to visualize different tissue types within a fused 3D metastructure. The tissue grouping matched the 2D histology applied only on 1 slice, thus extending the histology labelling in 3D. Specifically, in all samples, we could separate and map 2 types of regenerated bone, calcified tissue, soft tissues, and/or fat and marrow space. Remarkably, μMRI and μCT alone were not able to separate the 2 types of regenerated bone. Finally, we computed volumes of each tissue in the 3D metastructures, which might be exploited by quantitative simulation. The 3D metastructure obtained through our pipeline represents a first step to bridge the gap between the quality of information obtained from 2D optical microscopy and the 3D mapping of the bone tissue heterogeneity and could allow researchers and clinicians to non-destructively characterize and follow-up bone regeneration., (Copyright © 2017 John Wiley & Sons, Ltd.)

The analysis of the α-synuclein (aS) aggregation process, which is involved in Parkinson's disease etiopathogenesis, and of the structural feature of the resulting amyloid fibrils may shed light on the relationship between the structure of aS aggregates and their toxicity. This may be considered a paradigm of the ground work needed to tackle the molecular basis of all the protein-aggregation-related diseases. With this aim, we used chemical and physical dissociation methods to explore the structural organization of wild-type aS fibrils. High pressure (in the kbar range) and alkaline pH were used to disassemble fibrils to collect information on the hierarchic pathway by which distinct β-sheets sequentially unfold using the unique possibility offered by high-pressure Fourier transform infrared spectroscopy. The results point toward the formation of kinetic traps in the energy landscape of aS fibril disassembly and the presence of transient partially folded species during the process. Since we found that the dissociation of wild-type aS fibrils by high pressure is reversible upon pressure release, the disassembled molecules likely retain structural information that favors fibril reformation. To deconstruct the role of the different regions of aS sequence in this process, we measured the high-pressure dissociation of amyloids formed by covalent chimeric dimers of aS (syn-syn) and by the aS deletion mutant that lacks the C-terminus, i.e., aS (1-99). The results allowed us to single out the role of dimerization and that of the C-terminus in the complete maturation of fibrillar aS., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In the large class of molecules that maintain protein homeostasis, called molecular chaperones, chaperonins constitute a subclass that specifically assist the correct folding of newly synthesized proteins. Among them, Hsp60 is composed of a double heptameric ring structure with a large central cavity where the unfolded protein binds via hydrophobic interactions and is supported, in this function, by the co-chaperonin Hsp10. Hsp60 is typically located in the mitochondria, but in some pathological situations, such as cancers and chronic inflammatory diseases, Hsp60 accumulates in the cytoplasm. In these cases, cytoplasmatic Hsp60 is a mixture of mitochondrial Hsp60 secreted from mitochondria upon stress, and its precursor, called naïve Hsp60, never entered into the organella. The difference between the naïve and mitochondrial Hsp60s resides in the absence of the mitochondrial import signal (MIS) in the mitochondrial form, but information on their different structure and stability is still lacking. We present here a study on the stability against a chemical denaturant, of the different cytoplasmic Hsp60 species. By combining Circular Dichroism and Small Angle X-ray Scattering as experimental biophysical techniques to investigate Hsp60, we find that naïve and mitochondrial Hsp60 (mtHsp60) forms differ in their stability. Furthermore, specific responses from the two forms are discussed in terms of the biological environment they are working in, thus opening new questions on their biological function., (Copyright © 2017 Elsevier B.V. All rights reserved.)

α-synuclein amyloid fibrils are found in surviving neurons of Parkinson's disease affected patients, but the role they play in the disease development is still under debate. A growing number of evidences points to soluble oligomers as the major cytotoxic species, while insoluble fibrillar aggregates could even play a protection role. In this work, we investigate α-synuclein fibrils dissociation induced at high pressure by means of Small Angle X-ray Scattering and Fourier Transform Infrared Spectroscopy. Fibrils were produced from wild type α-synuclein and two familial mutants, A30P and A53T. Our results enlighten the different reversible nature of α-synuclein fibrils fragmentation at high pressure and suggest water excluded volumes presence in the fibrils core. Wild type and A30P species stabilized at high pressure are highly amyloidogenic and quickly re-associate into fibrils upon decompression, while A53T species shows a partial reversibility of the process likely due to the presence of an intermediate oligomeric state stabilized at high pressure. The amyloid fibrils dissociation process is here suggested to be associated to a negative activation volume, supporting the notion that α-synuclein fibrils are in a high-volume and high-compressibility state and hinting at the presence of a hydration-mediated activated state from which dissociation occurs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Proteins in living organisms exist in complex aqueous solutions or embedded in membranes. In solution, proteins are surrounded by a tightly bound hydration layer, which is more ordered and less mobile than bulk water. As a consequence, water plays a major role in controlling protein structure stability, conformational flexibility, dynamics, and functionality, but it also appears that protein surface regulates the structuring of the surrounding water. The presence of cosolvents can modify the hydration layer characteristics and then the whole protein structural and dynamical properties. Because cytoplasm or biological liquids are complex solutions, the knowledge of the solvation shell characteristics in mixed solvents should be considered as a crucial step in describing biological processes at molecular level. This review reports on recent studies on the structural and thermodynamic properties of model proteins dissolved in binary solvent mixtures by small-angle neutron scattering (SANS) and differential scanning microcalorimetry (DSC) techniques. We will show that contrast variation SANS experiments allow to acquire a direct knowledge of both protein structure and protein solvation shell (in terms of low-resolution shape and solvent/cosolvent composition), while DSC experiments provide information on all the relevant thermodynamic properties. We will focus on two main points. First, an extended description of the thermodynamic model used to define the equilibria between water and cosolvent molecules in the protein solvation shell will be presented. Second, the determination of the peculiar characteristics of the protein solvation layer, which will be illustrated by considering different systems. As a conclusion, we will show that the investigation of structure and thermodynamics of proteins in binary aqueous mixtures is an important way to understand the role of hydration in protein stability and activity.

In the last decades, the study of the mechanisms inducing amyloid fibril formation has involved several experimental and theoretical biophysical approaches. Many efforts have been made by scientist at the borderline between biology, chemistry, biochemistry and physics in order to understand why and in which way a protein starts its amyloidogenic pattern. This fundamental research issue is evolving in parallel to the development of drugs and inhibitors able to modify protein self assembly towards amyloid fibrils. Small angle xray and neutron scattering experiments represent suitable methods to investigate protein amyloidogenesis and the possible effects of inhibitors: they are in-solution techniques, require low amount of sample and their time-resolution makes it possible to follow aggregation pattern. In this paper we review small angle x-ray and neutron scattering studies dedicated to investigate amyloid β peptide and α-synuclein, related to Alzheimer`s and Parkinson`s diseases, respectively, together with some other studies that introduced innovative models to describe with small angle scattering techniques amyloid fibrillation processes.

Human Hsp60 chaperonin and its bacterial homolog GroEL, in association with the corresponding co-chaperonins Hsp10 and GroES, constitute important chaperone systems promoting the proper folding of several mitochondrial proteins. Hsp60 is also currently described as a ubiquitous molecule with multiple roles both in health conditions and in several diseases. Naïve Hsp60 bearing the mitochondrial import signal has been recently demonstrated to present different oligomeric organizations with respect to GroEL, suggesting new possible physiological functions. Here we present a combined investigation with circular dichroism and small-angle X-ray scattering of structure, self-organization, and stability of naïve Hsp60 in solution in comparison with bacterial GroEL. Experiments have been performed in different concentrations of guanidine hydrochloride, monitoring the dissociation of tetradecamers into heptamers and monomers, until unfolding. GroEL is proved to be more stable with respect to Hsp60, and the unfolding free energy as well as its dependence on denaturant concentration is obtained., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Neuronal membrane damage is related to the early impairments appearing in Alzheimer's disease due to the interaction of the amyloid β-peptide (Aβ) with the phospholipid bilayer. In particular, the ganglioside GM1, present with cholesterol in lipid rafts, seems to be able to initiate Aβ aggregation on membrane. We studied the thermodynamic and structural effects of the presence of GM1 on the interaction between Aβ and liposomes, a good membrane model system. Isothermal Titration Calorimetry highlighted the importance of the presence of GM1 in recruiting monomeric Aβ toward the lipid bilayer. Light and Small Angle X-ray Scattering revealed a different pattern for GM1 containing liposomes, both before and after interaction with Aβ. The results suggest that the interaction with GM1 brings to insertion of Aβ in the bilayer, producing a structural perturbation down to the internal layers of the liposome, as demonstrated by the obtained electron density profiles., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Septins comprise a family of proteins involved in a variety of cellular processes and related to several human pathologies. They are constituted by three structural domains: the N- and C-terminal domains, highly variable in length and composition, and the central domain, involved in the guanine nucleotide (GTP) binding. Thirteen different human septins are known to form heterogeneous complexes or homofilaments, which are stabilized by specific interactions between the different interfaces present in the domains. In this work, we have investigated by in-solution small-angle x-ray scattering the structural and thermodynamic properties of a human septin 3 construct, SEPT3-GC, which contains both of both interfaces (G and NC) responsible for septin-septin interactions. In order to shed light on the role of these interactions, small-angle x-ray scattering measurements were performed in a wide range of temperatures, from 2 up to 56°C, both with and without a nonhydrolysable form of GTP (GTPγS). The acquired data show a temperature-dependent coexistence of monomers, dimers, and higher-order aggregates that were analyzed using a global fitting approach, taking into account the crystallographic structure of the recently reported SEPT3 dimer, PDB:3SOP. As a result, the enthalpy, entropy, and heat capacity variations that control the dimer-monomer dissociation equilibrium in solution were derived and GTPγS was detected to increase the enthalpic stability of the dimeric species. Moreover, a temperature increase was observed to induce dissociation of SEPT3-GC dimers into monomers just preceding their reassembling into amyloid aggregates, as revealed by the Thioflavin-T fluorescence assays., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

It has been established that Hsp60 can accumulate in the cytosol in various pathological conditions, including cancer and chronic inflammatory diseases. Part or all of the cytosolic Hsp60 could be naïve, namely, bear the mitochondrial import signal (MIS), but neither the structure nor the in solution oligomeric organization of this cytosolic molecule has still been elucidated. Here we present a detailed study of the structure and self-organization of naïve cytosolic Hsp60 in solution. Results were obtained by different biophysical methods (light and X ray scattering, single molecule spectroscopy and hydrodynamics) that all together allowed us to assay a wide range of concentrations of Hsp60. We found that Naïve Hsp60 in aqueous solution is assembled in very stable heptamers and tetradecamers at all concentrations assayed, without any trace of monomer presence.

Many research topics in the fields of condensed matter and the life sciences are based on small-angle X-ray and neutron scattering techniques. With the current rapid progress in source brilliance and detector technology, high data fluxes of ever-increasing quality are produced. In order to exploit such a huge quantity of data and richness of information, wider and more sophisticated approaches to data analysis are needed. Presented here is GENFIT , a new software tool able to fit small-angle scattering data of randomly oriented macromolecular or nanosized systems according to a wide list of models, including form and structure factors. Batches of curves can be analysed simultaneously in terms of common fitting parameters or by expressing the model parameters via physical or phenomenological link functions. The models can also be combined, enabling the user to describe complex heterogeneous systems.

Methods: Combining small-angle X-ray and neutron scattering measurements with inelastic neutron scattering experiments, we investigated the impact of high hydrostatic pressure on the structure and dynamics of β-lactoglobulin (βLG) in aqueous solution., Background: βLG is a relatively small protein, which is predominantly dimeric in physiological conditions, but dissociates to monomer below about pH3., Results: High-pressure structural results show that the dimer-monomer equilibrium, as well as the protein-protein interactions, are only slightly perturbed by pressure, and βLG unfolding is observed above a threshold value of 3000bar. In the same range of pressure, dynamical results put in evidence a slowing down of the protein dynamics in the picosecond timescale and a loss of rigidity of the βLG structure. This dynamical behavior can be related to the onset of unfolding processes, probably promoted from water penetration in the hydrophobic cavity., General Significance: Results suggest that density and compressibility of water molecules in contact with the protein are key parameters to regulate the protein flexibility., (© 2013.)

Lipophilic guanilic derivatives (lipoGs) dissolved in organic solvents can undergo different self-assembly pathways based on different H-bonded motifs, e.g., the cyclic discrete G-quartet, which forms in the presence of alkali-metal ions, and the "infinite" tape-like G-ribbon observed in the absence of ions. Using in-solution small-angle X-ray scattering, we analyzed a series of lipoGs dissolved in cyclohexane in the presence of different salts. The formation of G-quartet based supramolecular aggregates has been confirmed, evidencing the coexistence equilibrium of octamers and noncovalent molecular nanowires (the so-called G-quadruplexes). By global fitting the scattering data, the concentration of the two kinds of particles as well as the nanowire length have been derived as a function of temperature for the different compounds and salts. The thermodynamic parameters show that the self-assembly aggregation process is enthalpy driven, while the observed enthalpy-entropy compensation suggests that similar stacking interactions control the self-assembly of the different compounds. However, the strength of the stacking interactions, and then the nanowire stability, depends on the nature of templating cations and on their capacity to fill the central cavity of quadruplexes, with the order Sr(+) < Na(+) ≲ K(+).