Molecular structural dynamics in water-ethanol mixtures: Spectroscopy with polarized neutrons simultaneously accessing collective and self-diffusion.

Binary mixtures of water with lower alcohols display non-linear phase behaviors upon mixing, which are attributed to potential cluster formation at the molecular level. Unravelling such elusive structures requires investigation of hydrogen-bonding sub-nanosecond dynamics. We employ high-resolution neutron time-of-flight spectroscopy with polarization analysis in combination with selective deuteration to study the concentration-dependent structural dynamics in the water rich part of the phase diagram of water-ethanol mixtures. This method enables simultaneous access to atomic correlations in space and time and allows us to separate spatially incoherent scattering probing self-diffusion of the ethanol fraction from the coherent scattering probing collective diffusion of the water network as a whole. Our observations indicate an enhanced rigidity of the hydrogen bond network at the mesoscopic length scale compared to the molecular scale as the ethanol fraction increases, which is consistent with the hypothesis of clusters.

The Onset of Molecule-Spanning Dynamics in Heat Shock Protein Hsp90.

Protein dynamics have been investigated on a wide range of time scales. Nano- and picosecond dynamics have been assigned to local fluctuations, while slower dynamics have been attributed to larger conformational changes. However, it is largely unknown how fast (local) fluctuations can lead to slow global (allosteric) changes. Here, fast molecule-spanning dynamics on the 100 to 200 ns time scale in the heat shock protein 90 (Hsp90) are shown. Global real-space movements are assigned to dynamic modes on this time scale, which is possible by a combination of single-molecule fluorescence, quasi-elastic neutron scattering and all-atom molecular dynamics (MD) simulations. The time scale of these dynamic modes depends on the conformational state of the Hsp90 dimer. In addition, the dynamic modes are affected to various degrees by Sba1, a co-chaperone of Hsp90, depending on the location within Hsp90, which is in very good agreement with MD simulations. Altogether, this data is best described by fast molecule-spanning dynamics, which precede larger conformational changes in Hsp90 and might be the molecular basis for allostery. This integrative approach provides comprehensive insights into molecule-spanning dynamics on the nanosecond time scale for a multi-domain protein.

Signature of functional enzyme dynamics in quasielastic neutron scattering spectra: The case of phosphoglycerate kinase.

We present an analysis of high-resolution quasi-elastic neutron scattering spectra of phosphoglycerate kinase which elucidates the influence of the enzymatic activity on the dynamics of the protein. We show that in the active state the inter-domain motions are amplified and the intra-domain asymptotic power-law relaxation ∝t-α is accelerated, with a reduced coefficient α. Employing an energy landscape picture of protein dynamics, this observation can be translated into a widening of the distribution of energy barriers separating conformational substates of the protein.

Biophysical Determinants for the Viscosity of Concentrated Monoclonal Antibody Solutions.

Monoclonal antibodies (mAbs) are particularly relevant for therapeutics due to their high specificity and versatility, and mAb-based drugs are hence used to treat numerous diseases. The increased patient compliance of self-administration motivates the formulation of products for subcutaneous (SC) administration. The associated challenge is to formulate highly concentrated antibody solutions to achieve a significant therapeutic effect, while limiting their viscosity and preserving their physicochemical stability. Protein-protein interactions (PPIs) are in fact the root cause of several potential problems concerning the stability, manufacturability, and delivery of a drug product. The understanding of macroscopic viscosity requires an in-depth knowledge on protein diffusion, PPIs, and self-association/aggregation. Here, we study the self-diffusion of different mAbs of the IgG1 subtype in aqueous solution as a function of the concentration and temperature by quasi-elastic neutron scattering (QENS). QENS allows us to probe the short-time self-diffusion of the molecules and therefore to determine the hydrodynamic mAb cluster size and to gain information on the internal mAb dynamics. Small-angle neutron scattering (SANS) is jointly employed to probe structural details and to understand the nature and intensity of PPIs. Complementary information is provided by molecular dynamics (MD) simulations and viscometry, thus obtaining a comprehensive picture of mAb diffusion.

Effects of flexibility in coarse-grained models for bovine serum albumin and immunoglobulin G.

We construct a coarse-grained, structure-based, low-resolution, 6-bead flexible model of bovine serum albumin (BSA, PDB: 4F5S), which is a popular example of a globular protein in biophysical research. The model is obtained via direct Boltzmann inversion using all-atom simulations of a single molecule, and its particular form is selected from a large pool of 6-bead coarse-grained models using two suitable metrics that quantify the agreement in the distribution of collective coordinates between all-atom and coarse-grained Brownian dynamics simulations of solutions in the dilute limit. For immunoglobulin G (IgG), a similar structure-based 12-bead model has been introduced in the literature [Chaudhri et al., J. Phys. Chem. B 116, 8045 (2012)] and is employed here to compare findings for the compact BSA molecule and the more anisotropic IgG molecule. We define several modified coarse-grained models of BSA and IgG, which differ in their internal constraints and thus account for a variation of flexibility. We study denser solutions of the coarse-grained models with purely repulsive molecules (achievable by suitable salt conditions) and address the effect of packing and flexibility on dynamic and static behavior. Translational and rotational self-diffusivity is enhanced for more elastic models. Finally, we discuss a number of effective sphere sizes for the BSA molecule, which can be defined from its static and dynamic properties. Here, it is found that the effective sphere diameters lie between 4.9 and 6.1 nm, corresponding to a relative spread of about ±10% around a mean of 5.5 nm.

Diffusive Dynamics of Bacterial Proteome as a Proxy of Cell Death.

Temperature variations have a big impact on bacterial metabolism and death, yet an exhaustive molecular picture of these processes is still missing. For instance, whether thermal death is determined by the deterioration of the whole or a specific part of the proteome is hotly debated. Here, by monitoring the proteome dynamics of E. coli, we clearly show that only a minor fraction of the proteome unfolds at the cell death. First, we prove that the dynamical state of the E. coli proteome is an excellent proxy for temperature-dependent bacterial metabolism and death. The proteome diffusive dynamics peaks at about the bacterial optimal growth temperature, then a dramatic dynamical slowdown is observed that starts just below the cell's death temperature. Next, we show that this slowdown is caused by the unfolding of just a small fraction of proteins that establish an entangling interprotein network, dominated by hydrophobic interactions, across the cytoplasm. Finally, the deduced progress of the proteome unfolding and its diffusive dynamics are both key to correctly reproduce the E. coli growth rate.

Role of fast dynamics in the complexation of G-quadruplexes with small molecules.

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.

Resolving molecular diffusion and aggregation of antibody proteins with megahertz X-ray free-electron laser pulses.

X-ray free-electron lasers (XFELs) with megahertz repetition rate can provide novel insights into structural dynamics of biological macromolecule solutions. However, very high dose rates can lead to beam-induced dynamics and structural changes due to radiation damage. Here, we probe the dynamics of dense antibody protein (Ig-PEG) solutions using megahertz X-ray photon correlation spectroscopy (MHz-XPCS) at the European XFEL. By varying the total dose and dose rate, we identify a regime for measuring the motion of proteins in their first coordination shell, quantify XFEL-induced effects such as driven motion, and map out the extent of agglomeration dynamics. The results indicate that for average dose rates below 1.06 kGy µs-1 in a time window up to 10 µs, it is possible to capture the protein dynamics before the onset of beam induced aggregation. We refer to this approach as correlation before aggregation and demonstrate that MHz-XPCS bridges an important spatio-temporal gap in measurement techniques for biological samples.

Short-Time Transport Properties of Bidisperse Suspensions of Immunoglobulins and Serum Albumins Consistent with a Colloid Physics Picture.

The crowded environment of biological systems such as the interior of living cells is occupied by macromolecules with a broad size distribution. This situation of polydispersity might influence the dependence of the diffusive dynamics of a given tracer macromolecule in a monodisperse solution on its hydrodynamic size and on the volume fraction. The resulting size dependence of diffusive transport crucially influences the function of a living cell. Here, we investigate a simplified model system consisting of two constituents in aqueous solution, namely, of the proteins bovine serum albumin (BSA) and bovine polyclonal gamma-globulin (Ig), systematically depending on the total volume fraction and ratio of these constituents. From high-resolution quasi-elastic neutron spectroscopy, the separate apparent short-time diffusion coefficients for BSA and Ig in the mixture are extracted, which show substantial deviations from the diffusion coefficients measured in monodisperse solutions at the same total volume fraction. These deviations can be modeled quantitatively using results from the short-time rotational and translational diffusion in a two-component hard sphere system with two distinct, effective hydrodynamic radii. Thus, we find that a simple colloid picture well describes short-time diffusion in binary mixtures as a function of the mixing ratio and the total volume fraction. Notably, the self-diffusion of the smaller protein BSA in the mixture is faster than the diffusion in a pure BSA solution, whereas the self-diffusion of Ig in the mixture is slower than in the pure Ig solution.

Structure and diffusive dynamics of aspartate α-decarboxylase (ADC) liganded with D-serine in aqueous solution.

Incoherent neutron spectroscopy, in combination with dynamic light scattering, was used to investigate the effect of ligand binding on the center-of-mass self-diffusion and internal diffusive dynamics of Escherichia coli aspartate α-decarboxylase (ADC). The X-ray crystal structure of ADC in complex with the D-serine inhibitor was also determined, and molecular dynamics simulations were used to further probe the structural rearrangements that occur as a result of ligand binding. These experiments reveal that D-serine forms hydrogen bonds with some of the active site residues, that higher order oligomers of the ADC tetramer exist on ns-ms time-scales, and also show that ligand binding both affects the ADC internal diffusive dynamics and appears to further increase the size of the higher order oligomers.

High-resolution Neutron Spectroscopy to Study Picosecond-nanosecond Dynamics of Proteins and Hydration Water.

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state. Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer. The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into µm sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Strikingly Different Roles of SARS-CoV-2 Fusion Peptides Uncovered by Neutron Scattering.

Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.

Self-Diffusive Properties of the Intrinsically Disordered Protein Histatin 5 and the Impact of Crowding Thereon: A Combined Neutron Spectroscopy and Molecular Dynamics Simulation Study.

Intrinsically disordered proteins (IDPs) are proteins that, in comparison with globular/structured proteins, lack a distinct tertiary structure. Here, we use the model IDP, Histatin 5, for studying its dynamical properties under self-crowding conditions with quasi-elastic neutron scattering in combination with full atomistic molecular dynamics (MD) simulations. The aim is to determine the effects of crowding on the center-of-mass diffusion as well as the internal diffusive behavior. The diffusion was found to decrease significantly, which we hypothesize can be attributed to some degree of aggregation at higher protein concentrations, (≥100 mg/mL), as indicated by recent small-angle X-ray scattering studies. Temperature effects are also considered and found to, largely, follow Stokes-Einstein behavior. Simple geometric considerations fail to accurately predict the rates of diffusion, while simulations show semiquantitative agreement with experiments, dependent on assumptions of the ratio between translational and rotational diffusion. A scaling law that previously was found to successfully describe the behavior of globular proteins was found to be inadequate for the IDP, Histatin 5. Analysis of the MD simulations show that the width of the distribution with respect to diffusion is not a simplistic mirroring of the distribution of radius of gyration, hence, displaying the particular features of IDPs that need to be accounted for.

Multiscale relaxation dynamics and diffusion of myelin basic protein in solution studied by quasielastic neutron scattering.

We report an analysis of high-resolution quasielastic neutron scattering spectra from Myelin Basic Protein (MBP) in solution, comparing the spectra at three different temperatures (283, 303, and 323 K) for a pure D2O buffer and a mixture of D2O buffer with 30% of deuterated trifluoroethanol (TFE). Accompanying experiments with dynamic light scattering and Circular Dichroism (CD) spectroscopy have been performed to obtain, respectively, the global diffusion constant and the secondary structure content of the molecule for both buffers as a function of temperature. Modeling the decay of the neutron intermediate scattering function by the Mittag-Leffler relaxation function, ϕ(t) = Eα(-(t/τ)α) (0 < α < 1), we find that trifluoroethanol slows down the relaxation dynamics of the protein at 283 K and leads to a broader relaxation rate spectrum. This effect vanishes with increasing temperature, and at 323 K, its relaxation dynamics is identical in both solvents. These results are coherent with the data from dynamic light scattering, which show that the hydrodynamic radius of MBP in TFE-enriched solutions does not depend on temperature and is only slightly smaller compared to the pure D2O buffer, except for 283 K, where it is much reduced. In accordance with these observations, the CD spectra reveal that TFE induces essentially a partial transition from ß-strands to α-helices, but only a weak increase in the total secondary structure content, leaving about 50% of the protein unfolded. The results show that MBP is for all temperatures and in both buffers an intrinsically disordered protein and that TFE essentially induces a reduction in its hydrodynamic radius and its relaxation dynamics at low temperatures.

Ligand Dynamics in Nanocrystal Solids Studied with Quasi-Elastic Neutron Scattering.

Nanocrystal surfaces are commonly populated by organic ligands, which play a determining role in the optical, electronic, thermal, and catalytic properties of the individual nanocrystals and their assemblies. Understanding the bonding of ligands to nanocrystal surfaces and their dynamics is therefore important for the optimization of nanocrystals for different applications. In this study, we use temperature-dependent, quasi-elastic neutron scattering (QENS) to investigate the dynamics of different surface bound alkanethiols in lead sulfide nanocrystal solids. We select alkanethiols with mono- and dithiol terminations, as well as different backbone types and lengths. QENS spectra are collected both on a time-of-flight spectrometer and on a backscattering spectrometer, allowing us to investigate ligand dynamics in a time range from a few picoseconds to nanoseconds. Through model-based analysis of the QENS data, we find that ligands can either (1) precess around a central axis, while simultaneously rotating around their own molecular axis, or (2) only undergo uniaxial rotation with no precession. We establish the percentage of ligands undergoing each type of motion, the average relaxation times, and activation energies for these motions. We determine, for example, that dithiols which link facets of neighboring nanocrystals only exhibit uniaxial rotation and that longer ligands have higher activation energies and show smaller opening angles of precession due to stronger ligand-ligand interactions. Generally, this work provides insight into the arrangement and dynamics of ligands in nanocrystal solids, which is key to understanding their mechanical and thermal properties, and, more generally, highlights the potential of QENS for studying ligand behavior.

Molecular Flexibility of Antibodies Preserved Even in the Dense Phase after Macroscopic Phase Separation.

Antibody therapies are typically based on high-concentration formulations that need to be administered subcutaneously. These conditions induce several challenges, inter alia a viscosity suitable for injection, sufficient solution stability, and preservation of molecular function. To obtain systematic insights into the molecular factors, we study the dynamics on the molecular level under strongly varying solution conditions. In particular, we use solutions of antibodies with poly(ethylene glycol), in which simple cooling from room temperature to freezing temperatures induces a transition from a well-dispersed solution into a phase-separated and macroscopically arrested system. Using quasi-elastic neutron scattering during in situ cooling ramps and in prethermalized measurements, we observe a strong decrease in antibody diffusion, while internal flexibility persists to a significant degree, thus ensuring the movement necessary for the preservation of molecular function. These results are relevant for a more dynamic understanding of antibodies in high-concentration formulations, which affects the formation of transient clusters governing the solution viscosity.

Temperature and salt controlled tuning of protein clusters.

The formation of molecular assemblies in protein solutions is of strong interest both from a fundamental viewpoint and for biomedical applications. While ordered and desired protein assemblies are indispensable for some biological functions, undesired protein condensation can induce serious diseases. As a common cofactor, the presence of salt ions is essential for some biological processes involving proteins, and in aqueous suspensions of proteins can also give rise to complex phase diagrams including homogeneous solutions, large aggregates, and dissolution regimes. Here, we systematically study the cluster formation approaching the phase separation in aqueous solutions of the globular protein BSA as a function of temperature (T), the protein concentration (cp) and the concentrations of the trivalent salts YCl3 and LaCl3 (cs). As an important complement to structural, i.e. time-averaged, techniques we employ a dynamical technique that can detect clusters even when they are transient on the order of a few nanoseconds. By employing incoherent neutron spectroscopy, we unambiguously determine the short-time self-diffusion of the protein clusters depending on cp, cs and T. We determine the cluster size in terms of effective hydrodynamic radii as manifested by the cluster center-of-mass diffusion coefficients D. For both salts, we find a simple functional form D(cp, cs, T) in the parameter range explored. The calculated inter-particle attraction strength, determined from the microscopic and short-time diffusive properties of the samples, increases with salt concentration and temperature in the regime investigated and can be linked to the macroscopic behavior of the samples.

Zinc determines dynamical properties and aggregation kinetics of human insulin.

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

Impact of Sucrose as Osmolyte on Molecular Dynamics of Mouse Acetylcholinesterase.

The enzyme model, mouse acetylcholinesterase, which exhibits its active site at the bottom of a narrow gorge, was investigated in the presence of different concentrations of sucrose to shed light on the protein and water dynamics in cholinesterases. The study was conducted by incoherent neutron scattering, giving access to molecular dynamics within the time scale of sub-nano to nanoseconds, in comparison with molecular dynamics simulations. With increasing sucrose concentration, we found non-linear effects, e.g., first a decrease in the dynamics at 5 wt% followed by a gain at 10 wt% sucrose. Direct comparisons with simulations permitted us to understand the following findings: at 5 wt%, sugar molecules interact with the protein surface through water molecules and damp the motions to reduce the overall protein mobility, although the motions inside the gorge are enhanced due to water depletion. When going to 10 wt% of sucrose, some water molecules at the protein surface are replaced by sugar molecules. By penetrating the protein surface, they disrupt some of the intra-protein contacts, and induce new ones, creating new pathways for correlated motions, and therefore, increasing the dynamics. This exhaustive study allowed for an explanation of the detail interactions leading to the observed non-linear behavior.

Lipid Dynamics in Membranes Slowed Down by Transmembrane Proteins.

Lipids and proteins, as essential components of biological cell membranes, exhibit a significant degree of freedom for different kinds of motions including lateral long-range mobility. Due to their interactions, they not only preserve the cellular membrane but also contribute to many important cellular functions as e.g., signal transport or molecular exchange of the cell with its surrounding. Many of these processes take place on a short time (up to some nanoseconds) and length scale (up to some nanometers) which is perfectly accessible by quasielastic neutron scattering (QENS) experiments and molecular dynamics (MD) simulations. In order to probe the influence of a peptide, a transmembrane sequence of the transferrin receptor (TFRC) protein, on the dynamics of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) large unilamellar vesicles (LUVs) on a nanosecond time scale, high-resolution QENS experiments and complementary MD simulations have been utilized. By using different scattering contrasts in the experiment (chain-deuterated lipids and protonated lipids, respectively), a model could be developed which allows to examine the lipid and peptide dynamics separately. The experimental results revealed a restricted lipid lateral mobility in the presence of the TFRC transmembrane peptides. Also the apparent self-diffusion coefficient of the lateral movement of the peptide molecules could be determined quantitatively for the probed short-time regime. The findings could be confirmed very precisely by MD simulations. Furthermore, the article presents an estimation for the radius of influence of the peptides on the lipid long-range dynamics which could be determined by consistently combining results from experiment and simulation.