Hypothesis Chondroitin sulphate (CS) is a linear polysaccharide typically found on the surface of cells and contributing to the structure of the extracellular matrix. Because of its proximity to the plasma membrane, the outermost cellular barrier, CS can interact with the phospholipids forming the structural scaffold of this cellular membrane. We hypothesise that the lipid composition of the plasma membrane, and specifically the exposure of phosphatidylserine (PS) lipids (an event that is detected in cancer cells and also associated to apoptosis and inflammation), affects the structural conformation of CS at the cell surface. Experiments We combined experimental data obtained with different techniques, i.e., quartz crystal microbalance with dissipation monitoring, neutron reflectometry and infrared spectroscopy, with molecular dynamics (MD) simulations to investigate the adsorption of CS at the surface of lipid bilayers prepared with either phosphatidylcholine (PC) lipids or a mixture of PC and PS lipids. Experiments were designed to identify the molecular groups that are involved in the CS-lipid interaction. Findings Our results indicate that CS adsorbs and remains stably attached to the lipid bilayer without PS lipids, due to stabilising interactions between the negatively charged sulphate groups on CS and positively charged choline groups within PC. The addition of POPS strongly reduces the CS-bilayer association: detecting experimentally CS chains attached to the bilayer was challenging, and the MD simulations suggest a weaker binding of CS to a PC-PS membrane.

G-quadruplexes (GQs), noncanonical DNA structures involved in gene regulation and genome stability, exhibit a high structural polymorphism that is strongly modulated by their solvation environment. Here we employ a dual-spectroscopy approach, combining ultraviolet resonance Raman (UVRR) scattering with circular dichroism (CD), to study GQ dilute solutions during thermal unfolding. UVRR scattering enhances the vibrational features of GQs, providing a method previously applied to other biosystems but novel for GQs in probing solute-solvent interactions. By analyzing the O-H stretching vibrational band, which reflects the hydrogen-bonded water network, we reveal how water molecules interact differently with two distinct GQ conformers, namely, hybrid and parallel. We demonstrate that coupling UVRR scattering with CD spectroscopy allows for the correlation of vibrational properties with secondary structural features of the solute, even in spectral regions dominated by solvent contributions. Expanding this approach to other GQs may offer deeper insights into the critical role of solvation in GQ stability and function.

The protein dynamical transition marks an increase in atomic mobility and the onset of anharmonic motions at a critical temperature (T d), which is considered relevant for protein functionality. This phenomenon is ubiquitous, regardless of protein composition, structure and biological function and typically occurs at large protein content, to avoid water crystallization. Recently, a dynamical transition has also been reported in non-biological macromolecules, such as poly(N-isopropyl acrylamide) (PNIPAM) microgels, bearing many similarities to proteins. While the generality of this phenomenon is well-established, the role of water in the transition remains a subject of debate. In this study, we use atomistic molecular dynamics (MD) simulations and elastic incoherent neutron scattering (EINS) experiments with selective deuteration to investigate the microscopic origin of the dynamical transition and distinguish water and PNIPAM roles. While a standard analysis of EINS experiments would suggest that the dynamical transition occurs in PNIPAM and water at a similar temperature, simulations reveal a different perspective, also qualitatively supported by experiments. From room temperature down to about 180 K, PNIPAM exhibits only modest changes of dynamics, while water, being mainly hydration water under the probed extreme confinement, significantly slows down and undergoes a mode-coupling transition from diffusive to activated. Our findings therefore challenge the traditional view of the dynamical transition, demonstrating that it occurs in proximity of the water mode-coupling transition, shedding light on the intricate interplay between polymer and water dynamics.

Telomeric G-quadruplexes (G4s) are non-canonical DNA structures composed of TTAGGG repeats. They are extensively studied both as biomolecules key for genome stability and as promising building blocks and functional elements in synthetic biology and nanotechnology. This is why it is extremely important to understand how the interaction between G4s is affected by their topology. We used small-angle x-ray scattering to investigate the end-to-end stacking of antiparallel telomeric G-quadruplexes formed by the sequence AG3(T2AG3)3. To represent the experimental data, we developed a highly efficient coarse-grained fitting tool, which successfully described the samples as an equilibrium mixture of monomeric and dimeric G4 species. Our findings indicate that the antiparallel topology prevents the formation of long multimeric structures under self-crowding conditions, unlike the hybrid/parallel structures formed by the same DNA sequence. This result supports the idea that the stacking of monomeric G-quadruplexes is strongly affected by the presence of diagonal loops.

The behavior of liquids has always been a subject of strong interest and still presents many challenging issues. A paradigmatic example is the understanding of their atomic-scale collective dynamics. Experimentally accessible to inelastic neutron and X-ray scattering, this regime has recently disclosed an unexpectedly rich scenario made up of more than one collective excitation. However, experiments lack a fundamental piece of information since they cannot distinguish the longitudinal or transverse nature of the detected modes. Here we present an inelastic neutron scattering study of the atomic dynamics of water and sulfuric acid, two prototypes of hydrogen bonded liquids. We propose a novel method that allows the experimental determination of the nature of the observed modes, without resorting to any indirect information. The dynamic structure factors show a complex collective dynamics, with two propagating excitations that we describe in the framework of an interacting-modes model. The first high-energy excitation is usually assumed to be the prolongation of the longitudinal acoustic mode and is characterized by a strong fast sound, which in turn we ascribe to the interaction with the second low-energy mode. Our approach confirms the main longitudinal nature of the first mode and unambiguously identifies the transverse nature of the second one. Finally, the lifetime of the modes suggests a common origin for the second mode, possibly related to the presence of the hydrogen bond network.

The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1’ and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV.

G-quadruplexes (G4s) are helical four-stranded structures forming from guanine-rich nucleic acid sequences, which are thought to play a role in cancer development and malignant transformation. Most current studies focus on G4 monomers, yet under suitable and biologically relevant conditions, G4s undergo multimerization. Here, we investigate the stacking interactions and structural features of telomeric G4 multimers by means of a novel low-resolution structural approach that combines small-angle X-ray scattering (SAXS) with extremely coarse-grained (ECG) simulations. The degree of multimerization and the strength of the stacking interaction are quantitatively determined in G4 self-assembled multimers. We show that self-assembly induces a significant polydispersity of the G4 multimers with an exponential distribution of contour lengths, consistent with a step-growth polymerization. On increasing DNA concentration, the strength of the stacking interaction between G4 monomers increases, as well as the average number of units in the aggregates. We utilized the same approach to explore the conformational flexibility of a model single-stranded long telomeric sequence. Our findings indicate that its G4 units frequently adopt a beads-on-a-string configuration. We also observe that the interaction between G4 units can be significantly affected by complexation with benchmark ligands. The proposed methodology, which identifies the determinants that govern the formation and structural flexibility of G4 multimers, may be an affordable tool aiding in the selection and design of drugs that target G4s under physiological conditions.

Telomeric G-quadruplexes (G4s) are promising targets in the design and development of anticancer drugs. Their actual topology depends on several factors, resulting in structural polymorphism. In this study, we investigate how the fast dynamics of the telomeric sequence AG(3)(TTAG(3))(3) (Tel22) depends on the conformation. By using Fourier transform Infrared spectroscopy, we show that, in the hydrated powder state, Tel22 adopts parallel and mixed antiparallel/parallel topologies in the presence of K+ and Na+ ions, respectively. These conformational differences are reflected in the reduced mobility of Tel22 in Na+ environment in the sub-nanosecond timescale, as probed by elastic incoherent neutron scattering. These findings are consistent with the G4 antiparallel conformation being more stable than the parallel one, possibly due to the presence of ordered hydration water networks. In addition, we study the effect of Tel22 complexation with BRACO19 ligand. Despite the quite similar conformation in the complexed and uncomplexed state, the fast dynamics of Tel22-BRACO19 is enhanced compared to that of Tel22 alone, independently of the ions. We ascribe this effect to the preferential binding of water molecules to Tel22 against the ligand. The present results suggest that the effect of polymorphism and complexation on the G4 fast dynamics is mediated by hydration water.

G-quadruplexes (G4s) formed by the human telomeric sequence AG3 (TTAG3)3 (Tel22) play a key role in cancer and aging. We combined elastic incoherent neutron scattering (EINS) and quasielastic incoherent neutron scattering (QENS) to characterize the internal dynamics of Tel22 G4s and to assess how it is affected by complexation with two standard ligands, Berberine and BRACO19. We show that the interaction with the two ligands induces an increase of the overall mobility of Tel22 as quantified by the mean squared displacements (MSD) of hydrogen atoms. At the same time, the complexes display a lower stiffness than G4 alone. Two different types of motion characterize the G4 nanosecond timescale dynamics. Upon complexation, an increasing fraction of G4 atomic groups participate in this fast dynamics, along with an increase in the relevant characteristic length scales. We suggest that the entropic contribution to the conformational free energy of these motions might be crucial for the complexation mechanisms.

G-quadruplexes (G4s) are helical four-stranded structures forming from guanine-rich nucleic acid sequences, which are thought to play a role in cancer development and malignant transformation. Most current studies focus on G4 monomers, yet under suitable and biologically relevant conditions G4s undergo multimerization. Here, we address the structural, conformational and energetic features of G4 multimers formed in solutions by the human telomere sequence. A novel multi-technique approach is used combining Small Angle X-ray Scattering (SAXS) and circular dichroism experiments with coarse-grained simulations, to provide quantitative information about large-scale structural features and the stability of G4 multimers. The latter show a significant polydispersity with an exponential distribution of contour lengths, suggesting a step-growth polymerization. On increasing DNA concentration, the strength of G4 stacking interaction increases, as well as the number of the units in the aggregates, with dimers and trimers as the most probable forms. At the same time, a variation of G4 conformation is observed. Our findings provide a quantitative picture of human telomere G4 multimers, which must be accounted for to achieve a rational design of anticancer drugs targeting DNA structures.

Poly(N-isopropylacrylamide) (PNIPAM) is a synthetic polymer that is widely studied for its thermoresponsive character. However, recent works also reported evidence of a low temperature (protein-like) dynamical transition around 225 K in concentrated PNIPAM suspensions, independently of the polymer architecture, i.e., both for linear chains and for microgels. In this work, we investigate water-polymer interactions by extensive differential scanning calorimetry (DSC) measurements of both systems, in order to understand the effect of the different topological structures on the solution behaviour, in particular regarding crystallization and melting processes. In addition, we compare protiated and deuterated microgels, in both water and deuterated water. The DSC results are complemented by dynamic light scattering experiments, which confirm that the selective isotopic substitution differently affects the solution behaviour. Our findings highlight the important role played by the polymer architecture on the solution behaviour: indeed, microgels turn out to be more efficient confining agents, able to avoid water crystallization in a wider concentration range with respect to linear chains. Altogether, the present data will be valuable to interpret future low-temperature investigations of PNIPAM dispersions, particularly by neutron scattering experiments.

By means of elastic incoherent neutron scattering, we investigated how the addition of stabilizing cosolvents (glycerol and glucose) affects the dynamics of hydrated PNIPAM chains at the pico- and nanosecond time scale, where a low-temperature dynamical transition is observed. From the elastic intensities, the atomic mean square displacements of the PNIPAM samples were extracted using a global fitting procedure. Both the dynamical transition temperature T-d and the amplitude of the displacements are found to be strongly dependent on solvent composition. The close analogies between the dynamical transition of PNIPAM and that of biomolecules reveal PNIPAM as an excellent system for reproducing complex solvent-biopolymer interactions.

E. Gutsmiedl, F. Böhle, A. Frei, A. Maier, S. Paul, H. Schober, 3 and A. Orecchini 4 Technische Universität München,Physik Department, James Franck Str. 1, D-85747 Garching, Germany Institut Laue Langevin, 6 rue Jules Horrowitz, F-38042 Grenoble Cedex 9, France Universite Joseph Fourier, UFR de Physique, F-38042 Grenoble Cedex 9, France Dipartimento di Fisica, Universit di Perugia, I-06123 Perugia, and CNR-INFM CRS-Soft c/o Universit di Roma La Sapienza, I-00185 Roma, Italy. (Dated: April 26, 2021)

Combining elastic incoherent neutron scattering experiments at different resolutions with molecular dynamics simulations, we report the observation of a proteinlike dynamical transition in linear chains of poly(N-isopropylacrylamide). We identify the onset of the transition at a temperature T-d of about 225 K. Due to a global fit procedure, we find quantitative agreement between measured and calculated polymer mean-squared displacements at all temperatures and time resolutions. Our results confirm the generality of the dynamical transition in macromolecular systems in aqueous environments, independently of the internal polymer topology.

An overview is provided of the 15 neutron beam instruments making up the initial instrument suite of the European Spallation Source (ESS), and being made available to the neutron user community. The ESS neutron source consists of a high-power accelerator and target station, providing a unique long-pulse time structure of slow neutrons. The design considerations behind the time structure, moderator geometry and instrument layout are presented. The 15-instrument suite consists of two small-angle instruments, two reflectometers, an imaging beamline, two single-crystal diffractometers; one for macromolecular crystallography and one for magnetism, two powder diffractometers, and an engineering diffractometer, as well as an array of five inelastic instruments comprising two chopper spectrometers, an inverse-geometry single-crystal excitations spectrometer, an instrument for vibrational spectroscopy and a high-resolution backscattering spectrometer. The conceptual design, performance and scientific drivers of each of these instruments are described. All of the instruments are designed to provide breakthrough new scientific capability, not currently available at existing facilities, building on the inherent strengths of the ESS long-pulse neutron source of high flux, flexible resolution and large bandwidth. Each of them is predicted to provide world-leading performance at an accelerator power of 2 MW. This technical capability translates into a very broad range of scientific capabilities. The composition of the instrument suite has been chosen to maximise the breadth and depth of the scientific impact of the early years of the ESS, and provide a solid base for completion and further expansion of the facility.

Local and propagating vibrations occurring in DNA in the terahertz domain are essential for processes at the basis of cellular metabolism. Here we report the results of a study on the B-DNA terahertz dynamics where we combined high-resolution inelastic x-ray experiments and incoherent inelastic neutron scattering. By using two different high-hydration conditions we could study the effect of packing interactions between double helices, while by selecting Na and Cs counterions we could inspect how the mass loading affects the DNA low-frequency modes. The pattern of coherent excitation energies is well represented in terms of two branches, an acoustic-like one, with an associated propagation velocity of 3000 +/- 100 m/s, and the other almost dispersionless at similar to 2 meV. This picture is also supported by the vibrational density of states projected on the hydrogen atoms. The acoustic-like mode is assigned to DNA excitations, despite its intriguing similarity with the analogous acoustic mode of bulk water at low wave-vector transfers. We also provide evidence for the intrahelical optic-like character of the low-energy mode, that we ascribe to large scale relative motions of DNA sub-domains. (C) 2020 Elsevier B.V. All rights reserved.

Combining elastic incoherent neutron scattering and differential scanning calorimetry, we investigate the occurrence of the volume phase transition (VPT) in very concentrated poly-(N-isopropyl-acrylamide) (PNIPAM) microgel suspensions, from a polymer weight fraction of 30 wt. % up to dry conditions. Although samples are arrested at the macroscopic scale, atomic degrees of freedom are equilibrated and can be probed in a reproducible way. A clear signature of the VPT is present as a sharp drop in the mean square displacement of PNIPAM hydrogen atoms obtained by neutron scattering. As a function of concentration, the VPT gets smoother as dry conditions are approached, whereas the VPT temperature shows a minimum at about 43 wt. %. This behavior is qualitatively confirmed by calorimetry measurements. Molecular dynamics simulations are employed to complement experimental results and gain further insights into the nature of the VPT, confirming that it involves the formation of an attractive gel state between the microgels. Overall, these results provide evidence that the VPT in PNIPAM-based systems can be detected at different time- and length-scales as well as under overcrowded conditions.

By combined use of wide-angle X-ray scattering, thermo-gravimetric analysis, inelastic neutron scattering, density functional theory and density functional theory molecular dynamics simulations, we investigate the structure, dynamics and stability of the water wetting-layer in single-walled aluminogermanate imogolite nanotubes (SW Ge-INTs): an archetypal system for synthetically controllable and monodisperse nanoreactors. We demonstrate that the water wetting-layer is strongly bound and solid-like up to 300 K under atmospheric pressure, with dynamics markedly different from that of bulk water. Atomic-scale characterisation of the wetting-layer reveals organisation of the H2O molecules in a curved triangular sublattice stabilised by the formation of three H-bonds to the nanotube's inner surface, with covalent interactions sufficiently strong to promote energetically favourable decoupling of the H2O molecules in the adlayer. The evidenced changes in the local composition, structure, electrostatics and dynamics of the Ge-INT's inner surface upon the formation of the solid wetting-layer demonstrate solvent-mediated functionalisation of the nanotube's cavity at room temperature and pressure, suggesting new strategies for the design of nano-rectors towards potential control of chemical reactivity in nano-confined volumes.

Smart polymers as poly-N-isopropylacrylamide (PNIPAM) are well known due to their fast response to variations of parameters like temperature, pH, and pressure. [...]