Elastic Incoherent Neutron Scattering Research Articles

Molecular Dynamics of Lysozyme Amyloid Polymorphs Studied by Incoherent Neutron Scattering.

Lysozyme amyloidosis is a hereditary disease, which is characterized by the deposition of lysozyme amyloid fibrils in various internal organs. It is known that lysozyme fibrils show polymorphism and that polymorphs formed at near-neutral pH have the ability to promote more monomer binding than those formed at acidic pH, indicating that only specific polymorphs become dominant species in a given environment. This is likely due to the polymorph-specific configurational diffusion. Understanding the possible differences in dynamical behavior between the polymorphs is thus crucial to deepen our knowledge of amyloid polymorphism and eventually elucidate the molecular mechanism of lysozyme amyloidosis. In this study, molecular dynamics at sub-nanosecond timescale of two kinds of polymorphic fibrils of hen egg white lysozyme, which has long been used as a model of human lysozyme, formed at pH 2.7 (LP27) and pH 6.0 (LP60) was investigated using elastic incoherent neutron scattering (EINS) and quasi-elastic neutron scattering (QENS). Analysis of the EINS data showed that whereas the mean square displacement of atomic motions is similar for both LP27 and LP60, LP60 contains a larger fraction of atoms moving with larger amplitudes than LP27, indicating that the dynamical difference between the two polymorphs lies not in the averaged amplitude, but in the distribution of the amplitudes. Furthermore, analysis of the QENS data showed that the jump diffusion coefficient of atoms is larger for LP60, suggesting that the atoms of LP60 undergo faster diffusive motions than those of LP27. This study thus characterizes the dynamics of the two lysozyme polymorphs and reveals that the molecular dynamics of LP60 is enhanced compared with that of LP27. The higher molecular flexibility of the polymorph would permit to adjust its conformation more quickly than its counterpart, facilitating monomer binding.

Differences between calcium rich and depleted alpha-lactalbumin investigated by molecular dynamics simulations and incoherent neutron scattering.

We present a study comparing atomic motional amplitudes in calcium rich and depleted alpha-lactalbumin. The investigations were performed by elastic incoherent neutron scattering (EINS) and molecular dynamics (MD) simulations. As the variations were expected to be very small, three different hydration levels and timescales (instrumental resolutions) were measured. In addition, we used two models to extract the mean square displacements (MSDs) from the EINS data, one taking into account the motional heterogeneity of the MSD. At a timescale of several nanoseconds, small differences in the amplitudes between the calcium enriched and depleted alpha-lactalbumin are visible, whereas at lower timescales no changes can be concluded within the statistics. The results are compared to MD simulations at 280 and 300 K by extracting the MSDs of the trajectories in two separate ways: first by direct calculation, and second by a virtual neutron experiment using the same models as for the experimental data. We show that the simulated data give qualitatively similar results as the experimental data but quantitatively there are differences. Furthermore, the distribution of the MSDs in the simulations suggests that the inclusion of heterogeneity is reasonable for alpha-lactalbumin, but a bi-or trimodal approach may be sufficient.

Protein Backbone and Average Particle Dynamics in Reconstituted Discoidal and Spherical HDL Probed by Hydrogen Deuterium Exchange and Elastic Incoherent Neutron Scattering

Lipoproteins are supramolecular assemblies of proteins and lipids with dynamic characteristics critically linked to their biological functions as plasma lipid transporters and lipid exchangers. Among them, spherical high-density lipoproteins are the most abundant forms of high-density lipoprotein (HDL) in human plasma, active participants in reverse cholesterol transport, and associated with reduced development of atherosclerosis. Here, we employed elastic incoherent neutron scattering (EINS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to determine the average particle dynamics and protein backbone local mobility of physiologically competent discoidal and spherical HDL particles reconstituted with human apolipoprotein A-I (apoA-I). Our EINS measurements indicated that discoidal HDL was more dynamic than spherical HDL at ambient temperatures, in agreement with their lipid-protein composition. Combining small-angle neutron scattering (SANS) with contrast variation and MS cross-linking, we showed earlier that the most likely organization of the three apolipoprotein A-I (apoA-I) chains in spherical HDL is a combination of a hairpin monomer and a helical antiparallel dimer. Here, we corroborated those findings with kinetic studies, employing hydrogen-deuterium exchange mass spectrometry (HDX-MS). Many overlapping apoA-I digested peptides exhibited bimodal HDX kinetics behavior, suggesting that apoA-I regions with the same amino acid composition located on different apoA-I chains had different conformations and/or interaction environments.

Picosecond Dynamical Response to a Pressure-Induced Break of the Tertiary Structure Hydrogen Bonds in a Membrane Chromoprotein.

We used elastic incoherent neutron scattering (EINS) to find out if structural changes accompanying local hydrogen bond rupture are also reflected in global dynamical response of the protein complex. Chromatophore membranes from LH2-only strains of the photosynthetic bacterium Rhodobacter sphaeroides, with spheroidenone or neurosporene as the major carotenoids, were subjected to high hydrostatic pressure at ambient temperature. Optical spectroscopy conducted at high pressure confirmed rupture of tertiary structure hydrogen bonds. In parallel, we used EINS to follow average motions of the hydrogen atoms in LH2, which reflect the flexibility of this complex. A decrease of the average atomic mean square displacements of hydrogen atoms was observed up to a pressure of 5 kbar in both carotenoid samples due to general stiffening of protein structures, while at higher pressures a slight increase of the displacements was detected in the neurosporene mutant LH2 sample only. These data show a correlation between the local pressure-induced breakage of H-bonds, observed in optical spectra, with the altered protein dynamics monitored by EINS. The slightly higher compressibility of the neurosporene mutant sample shows that even subtle alterations of carotenoids are manifested on a larger scale and emphasize a close connection between the local structure and global dynamics of this membrane protein complex.

Influence of Enantiomeric Inhibitors on the Dynamics of Acetylcholinesterase Measured by Elastic Incoherent Neutron Scattering.

The enzyme acetylcholinesterase (AChE) is essential in humans and animals because it catalyzes the breakdown of the nerve-signaling substance acetylcholine. Small molecules that inhibit the function of AChE are important for their use as drugs in the, for example, symptomatic treatment of Alzheimer's disease. New and improved inhibitors are warranted, mainly because of severe side effects of current drugs. In the present study, we have investigated if and how two enantiomeric inhibitors of AChE influence the overall dynamics of noncovalent complexes, using elastic incoherent neutron scattering. A fruitful combination of univariate models, including a newly developed non-Gaussian model for atomic fluctuations, and multivariate methods (principal component analysis and discriminant analysis) was crucial to analyze the fine details of the data. The study revealed a small but clear increase in the dynamics of the inhibited enzyme compared to that of the noninhibited enzyme and contributed to the fundamental knowledge of the mechanisms of AChE-inhibitor binding valuable for the future development of inhibitors.

On the interpretation of the temperature dependence of the mean square displacement (MSD) of protein, obtained from the incoherent neutron scattering

A variety of models and hypothesis have been proposed to interpret the temperature dependence of the mean square displacement (MSD) of protein, that is obtained from the incoherent neutron scattering measurement, but a consensus seems yet to be achieved. The most controversial point is concerned with the physics behind the abrupt change of gradient of MSD plotted against temperature. The phenomenon is attributed to different physics by different authors, such as glass transition, alpha to beta transition, harmonic to anharmonic transition, coupling of protein and hydrated-water dynamics, and so forth. In the present paper, we propose a theory to analyse the elastic incoherent neutron scattering (EINS) data of aqueous solutions of protein, based on the generalized Langevin theory combined with the 3D-RISM/RISM equation [B. Kim and F. Hirata, J. Chem. Phys., 138, 054108 (2012)]. The theory gives closed equations for the elastic incoherent structure factor (EISF) and MSD as a function of temperature. Based on the theory, the abrupt change of the gradient of MSD in the temperature dependence is interpreted as an onset of “solvent-induced elasticity,” or “free energy elasticity,” with increasing temperature.

Thermodynamics of lipid multi-lamellar vesicles in presence of sterols at high hydrostatic pressure

We compared the effect of cholesterol at different concentration on the phase behaviour of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) multilamellar vesicles. We used pressure perturbation differential scanning calorimetry (PPC) that studies a system on the whole by giving access to relevant thermodynamic quantities, and elastic incoherent neutron scattering (EINS) that probes local motions of a system at the atomic level by allowing extraction of dynamical parameters. PPC revealed that the volume expansion coefficient of DMPC and DMPC/Cholesterol samples with 13 and 25 mol% cholesterol is a linear function of the heat capacity measured by differential scanning calorimetry. Neutron backscattering spectroscopy showed that the mean square displacements of H atoms do exhibit an increase with temperature and a decrease under increasing pressure. Cholesterol added at concentrations of 25 and 50 mol% led to suppression of the main phase transition. Taking advantage of these results, the present study aims (i) to show that calorimetry and EINS using the Bicout and Zaccai model equally permit to get access to thermodynamic quantities characterizing pure DMPC and DMPC/cholesterol mixtures, thus directly confirming the theoretical method, and (ii) to validate our approach as function of temperature and of pressure, as both are equally important and complementary thermodynamic variables.

Low-density lipoproteins investigated under high hydrostatic pressure by elastic incoherent neutron scattering

.Human low-density lipoprotein (LDL) is a highly complex nano-particle built up of various lipid classes and a single large protein moiety (apoB-100) owning essential physiological functions in the human body. Besides its vital role as a supplier of cholesterol and fat for peripheral tissues and cells, it is also a known key player in the formation of atherosclerosis. Due to these important roles in physiology and pathology the elucidation of structural and dynamical details is of great interest. In the current study we drew a broader picture of LDL dynamics using elastic incoherent neutron scattering (EINS) as a function of specified temperature and pressure points. We not only investigated a normolipidemic LDL sample, but also a triglyceride-rich and an oxidized one to mimic pathologic conditions as found under hyperlipidemic conditions or in atherosclerotic plaques, respectively. We could show that pressure has a significant effect on atomic motions in modified forms of LDL, whereas the normolipidemic sample seems to cope much better with high-pressure conditions irrespective of temperature. These findings might be explained by the altered lipid composition, which is either caused through elevated triglyceride content or modifications through lipid peroxidation.Graphical abstract

Contrasting dynamics of fragile and non-fragile polyalcohols through the glass, and dynamical, transitions: A comparison of neutron scattering and dielectric relaxation data for sorbitol and glycerol

BackgroundGlycerol and sorbitol are glass-forming hydrogen-bonded systems characterized by intriguing properties which make these systems very interesting also from the applications point of view. The goal of this work is to relate the hydrogen-bonded features, relaxation dynamics, glass transition properties and fragility of these systems, in particular to seek insight into their very different liquid fragilities. MethodsThe comparison between glycerol and sorbitol is carried out by collecting the elastic incoherent neutron scattering (EINS) intensity as a function of temperature and of the instrumental energy resolution. ResultsIntensity data vs temperature and resolution are analyzed in terms of thermal restraint and Resolution Elastic Neutron Scattering (RENS) approaches. ConclusionsThe number of OH groups, which are related to the connecting sites, is a significant parameter both in the glass transition and in the dynamical transition. On the other hand, the disordered nature of sorbitol is confirmed by the existence of different relaxation processes. General significanceFrom the applications point of view, glycerol and sorbitol have remarkable bioprotectant properties which make these systems useful in different technological and industrial fields. Furthermore, polyols are rich in glassforming liquid phenomenology and highly deserving of study in their own right. The comparison of EINS and calorimetric data on glycerol and sorbitol helps provide a connection between structural relaxation, dynamical transition, glass transition, and fragility. The evaluation of the inflection point in the elastic intensity behavior as a function of temperature and instrumental energy resolution provides a confirmation of the validity of the RENS approach.This article is part of a Special Issue entitled “Science for Life” Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo

Probing the Thermal Stability of Lysozyme in Crowded Environments: Tracking Lindemann Criterion

Our work focuses on determining the effect of crowded environment and different solvents on the thermal stability of the protein Lysozyme [1] placed in a dilute water solution, dehydrated protein powder, and in a protein powder glycerol solution, the latter two representing crowded environments.The ultimate goal of our work is to probe the validity of the Lindemann criterion for protein melting [2, 3]. We employ an enhanced sampling Molecular Dynamics technique, REST2 [4, 5], where mutually exchangeable protein replicas are simulated at different effective temperatures, achieved by rescaling the force-field potential energy terms. The simulations are paralleled with Elastic Incoherent Neutron Scattering experiments.We first estimate the in silico melting temperature of our systems and reconstruct the stability curves. Instructed by this information, we calculate the scaling of atomic fluctuation approaching melting. Our results show that the atomic fluctuations of different Lysozyme systems converge to similar values approaching the melting temperature, which agrees both with the experimental results [unpublished] and the Lindemann criterion [2, 3]. Furthermore, we show that the thermal stability of the hydrated Lysozyme is increased in the presence of crowders (powder) and even more so if glycerol is present in addition to the crowders. The molecular factors, excluded volume versus specific interactions, will be discussed as source of the stability shifts.[1] H.-X. Zhou, G. Rivas, A.P. Minton, Annu. Rev. Biophys. (2008) 37, 375-397.[2] C. Chakravarty, P.G. Debenedetti, F.H. Stillinger, J. Chem. Phys. (2007) 126, 204508.[3] Y. Zhou, D. Vitkup, M. Karplus, J. Mol. Biol. (1999) 285, 1371-1375[4] L. Wang, R.A. Friesner, B.J. Berne, J. Phys. Chem. B (2011) 115, 9431-9438.[5] G. Stirnemann and F. Sterpone, submitted to J. Chem. Theory Comput. (2015).

Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.

Influence of Pressure and Crowding on the Sub-Nanosecond Dynamics of Globular Proteins

The influence of high hydrostatic pressure on the internal sub-nanosecond dynamics of highly concentrated lysozyme in aqueous solutions was studied by elastic incoherent neutron scattering (EINS) up to pressures of 4 kbar. We have found, with increasing pressure, a reduction in the dynamics of H atoms of folded lysozyme, suggesting a loss in protein mobility that follows a change in the local energy landscape upon the increase in packing density. Moreover, the amplitude of the protein fluctuations depends drastically on the protein concentration, and protein structural and interaction parameters as well as the dynamical properties are affected by pressure in a nonlinear way. A significant reduction of the mean squared displacement of H atoms occurs already at rather low pressures of a few hundred bars for lysozyme in bulk water solution. This trend is lifted at ∼2 kbar, which is probably due to a solvent-mediated effect. Conversely, for high protein concentrations (e.g., 160 mg mL(-1)), that is, under strong self-crowding conditions, as they are also encountered in the biological cell, strong restriction of the dynamics of protein motions takes place, reducing the mean squared displacement of H atoms by 60% and rendering its pressure dependence almost negligible. These results are also important for understanding the pressure stability of highly concentrated protein solutions in organisms thriving under hydrostatic pressure conditions such as in the deep sea, where pressures up to the kbar level are reached.

Pressure-induced molten globule state of human acetylcholinesterase: structural and dynamical changes monitored by neutron scattering.

We used small-angle neutron scattering (SANS) to study the effects of high hydrostatic pressure on the structure of human acetylcholinesterase (hAChE). At atmospheric pressure, our SANS results obtained on D11 at ILL (Grenoble, France) give a radius of gyration close to that calculated for a mixture of monomers, dimers and tetramers of the enzyme, suggesting a good agreement between hAChE crystal structure and its conformation in solution. Applying high pressure to the sample we found a global compression of about 11% of the enzyme up to a pressure of 900 bar and then again an extension up to 2.1 kbar indicating unfolding of the tertiary structure due to a molten globule (MG) state. On the other hand, we studied the influence of pressure up to 6 kbar on the dynamics of this enzyme, on the backscattering spectrometer IN13 at ILL. For the first time, we used elastic incoherent neutron scattering (EINS) to probe the differences between hAChE in its folded state (N), its high-pressure induced MG state and its unfolded state (U). Especially around the MG state at 1750 bar we found a significant increase in the dynamics, indicating a partial unfolding. A four-step-model is suggested to describe the changes in the protein.

Molecular mobility in Medicago truncatula seed during early stage of germination: Neutron scattering and NMR investigations

First hours of Medicago truncatula (MT) seeds germination were investigated using elastic incoherent neutron scattering (EINS) and nuclear magnetic resonance (NMR), to follow respectively how macromolecular motions and water mobility evolve when water permeates into the seed. From EINS results, it was shown that there is an increase in macromolecular mobility with the water uptake. Changes in NMR relaxation parameters reflected microstructural changes associated with the recovery of the metabolic processes. The EINS investigation of the effect of temperature on macromolecular motions showed that there is a relationship between the amount of water in the seeds and the effect of freezing–thawing cycle. The NMR relaxometry results obtained at 253K allowed establishing possible link between the freezing of water molecules tightly bound to macromolecules and their drastic motion restriction around 250K, as observed with EINS at the highest water content.

Thermal Analysis on Bioprotectant Disaccharides by Elastic Incoherent Neutron Scattering

In this work the attention is focused on the thermal properties of trehalose, a glass-forming disaccharide which is very effective as a shelf-life extending compound in food industry. A wavelet analysis of Elastic Incoherent Neutron Scattering (EINS) intensity data as a function of the exchanged wavevector on trehalose and its homologous, maltose and sucrose, is presented. This analysis, which allows to highlight the correlation between the signal and the set of the scaled and translated mother functions, reveals the existence of different kinds of protons dynamics which cover different spatial scales. Differently from previous analyses, it emerges that the energy distribution as a function of the exchanged wavevector for trehalose mixture is less sensitive to temperature changes. Furthermore, the application of a new fitting model to the partial and global EINS intensity data as a function of temperature allows to put into evidence both the different wavevector dependence of the system relaxation times, extracted from the intensity vs temperature inflection point, and the higher thermal resistance of trehalose in respect to the other investigated disaccharides.

Elastic incoherent neutron scatterings wavevector and thermal analysis on glass-forming homologous disaccharides

A wavelet analysis of Elastic Incoherent Neutron Scattering (EINS) intensity data collected, as a function of wavevector, on three glass forming systems, i.e. on the homologous disaccharides trehalose, maltose and sucrose, is presented. The wavelet transform allows to explore the wavevector behavior of the scattered intensity at different scales highlighting the correlation between the signal and the set of the scaled and translated mother functions. The analysis performed in the wavevector range of Q=0.28Å−1÷4.28Å−1 reveals the existence of different kinds of protons dynamics which cover different spatial scales. It emerges that, differently from previous analyses, for trehalose the intensity data at all the investigated temperature values are constantly lower and sharper in respect to those of maltose and sucrose, giving rise to a global spectral density along the wavevector range markedly less extended. Furthermore, the application of an interpretative model for the partial and global EINS intensity temperature behavior allows to put into evidence both the different wavevector dependence of the system relaxation times, extracted from the intensity vs. temperature inflection point, and the higher thermal restrain of trehalose in respect to the other two homologous disaccharides.

Investigations of homologous disaccharides by elastic incoherent neutron scattering and wavelet multiresolution analysis

In the present paper the results of a wavevector and thermal analysis of Elastic Incoherent Neutron Scattering (EINS) data collected on water mixtures of three homologous disaccharides through a wavelet approach are reported. The wavelet analysis allows to compare both the spatial properties of the three systems in the wavevector range of Q=0.27Å−1÷4.27Å−1. It emerges that, differently from previous analyses, for trehalose the scalograms are constantly lower and sharper in respect to maltose and sucrose, giving rise to a global spectral density along the wavevector range markedly less extended. As far as the thermal analysis is concerned, the global scattered intensity profiles suggest a higher thermal restrain of trehalose in respect to the other two homologous disaccharides.

Hydration Water Dynamics in Tricalcium Silicate Pastes by Time-Resolved Incoherent Elastic Neutron Scattering

The translational dynamics of the hydration water in tricalcium silicate, as well as the curing kinetics of the paste, have been investigated for the first time using neutron elastic backscattering measurements. A detailed analytical model has been formulated to extract the most important features of the calcium silicate curing process. As the curing time passes, water reacts with the tricalcium silicate, forming amorphous calcium silicate hydrate and crystalline calcium hydroxide. Concurrently, the water translational dynamics slows down due to the increased confinement in the developing matrix. The time evolution of the self-diffusion constant for the hydration water shows a decrease of 1 order of magnitude after two days of curing. The presented results agree with recent molecular dynamics simulations and 1H NMR results on similar systems.

Protein dynamics by neutron scattering: The protein dynamical transition and the fragile-to-strong dynamical crossover in hydrated lysozyme

In this work Elastic Incoherent Neutron Scattering (EINS) results on lysozyme water mixtures in absence and in presence of bioprotectant systems are presented. The EINS data have been collected by using the IN13 and the IN10 spectrometers at the Institut Laue-Langevin (ILL, Grenoble, France) allowing to evaluate the temperature behaviour of the mean square displacement and of the relaxation time for the investigated systems. The obtained experimental findings together with theoretical calculations allow to put into evidence the role played by the spectrometer resolution and to clarify the connexion between the registered protein dynamical transition, the system relaxation time, and the instrumental energy resolution.

Bioprotectant Effectiveness of Homologous Disaccharides by Elastic Incoherent Neutron Scattering

A new wavevector analysis performed by wavelet transform of Elastic Incoherent Neutron Scattering (EINS) data on three bioprotectant systems, i.e. on the homologous disaccharides trehalose, maltose and sucrose, is presented. The analysis allows to compare the spatial properties of the three systems in the wavevector range of , revealing the existence of different kinds of protons dynamics. It emerges that, differently from previous wavevector analyses, both the low and high wavevector contributions for trehalose, at all the investigated temperature values, are constantly lower and sharper, giving rise to a global energy distribution along the wavevector range markedly less extended. Furthermore an innovative model for the global EINS intensity temperature behavior is applied to put into evidence the higher thermal restrain for trehalose in respect to the other two homologous disaccharides. These findings give an account for the bioprotectant effectiveness of trehalose in the medical, pharmaceutical and cosmetic fields.