| 1. |
- Beck, Christian, et al.
(author)
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Following Protein Dynamics in Real Time during Crystallization
- 2019
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In: Crystal Growth and Design. - : American Chemical Society (ACS). - 1528-7483 .- 1528-7505. ; , s. 7036-7045
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Journal article (peer-reviewed)abstract
- The process of protein crystallization from aqueous protein solutions is still insufficiently understood. During macroscopic crystal formation, occurring often on time scales from a few hours to several days, protein dynamics evolves on the molecular level. Here, we present a proof of concept and a framework to observe this evolving diffusive dynamics on the pico- to nanosecond time scale, associated with cluster or precursor formation that ultimately results in emerging crystals. We investigated the model system of the protein β-lactoglobulin in D2O in the presence of ZnCl2, which induces crystallization by electrostatic bridges. First, the structural changes occurring during crystallization were followed by small-angle neutron scattering. Furthermore, we employed neutron backscattering and spin-echo spectroscopy to measure the ensemble-averaged self- and collective diffusion on nanosecond time scales of protein solutions with a kinetic time resolution on the order of 15 min. The experiments provide information on the increasing number fraction of immobilized proteins as well as on the diffusive motion of unbound proteins in an increasingly depleted phase. Simultaneously, information on the internal dynamics of the proteins is obtained.
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| 2. |
- Beck, Christian, et al.
(author)
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Neutron spectroscopy on protein solutions employing backscattering with an increased energy range
- 2019
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In: Physica B: Condensed Matter. - : Elsevier BV. - 0921-4526. ; 562, s. 31-35
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Journal article (peer-reviewed)abstract
- Novel cold neutron backscattering spectrometers contribute substantially to the understanding of the diffusive dynamics of proteins in dense aqueous suspensions. Such suspensions are fundamentally interesting for instance in terms of the so-called macromolecular crowding, protein cluster formation, gelation, and self-assembly. Notably, backscattering spectrometers with the highest flux can simultaneously access the center-of-mass diffusion of the proteins and the superimposed internal molecular diffusive motions. The nearly complete absence of protein-protein collisions on the accessible nanosecond observation time scale even in dense protein suspensions implies that neutron backscattering accesses the so-called short-time limit for the center-of-mass diffusion. This limit is particularly interesting in terms of a theoretical understanding by concepts from colloid physics. Here we briefly review recent progress in studying protein dynamics achieved with the latest generation of backscattering spectrometers. We illustrate this progress by the first data from a protein solution using the backscattering-and-time-of-flight option BATS on IN16B at the ILL and we outline future perspectives.
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| 3. |
- Beck, Christian, et al.
(author)
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Notes on Fitting and Analysis Frameworks for QENS Spectra of (Soft) Colloid Suspensions
- 2022
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In: EPJ Web of Conferences. - : EDP Sciences. - 2100-014X. ; 272, s. 01004-01004
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Journal article (peer-reviewed)abstract
- With continuously improving signal-to-noise ratios, a statistically sound analysis of quasi-elasticneutron scattering (QENS) spectra requires to fit increasingly complex models which poses several challenges.Simultaneous fits of the spectra for all recorded values of the momentum transfer become a standard approach.Spectrometers at spallation sources can have a complicated non-Gaussian resolution function which has to bedescribed most accurately. At the same time, to speed up the fitting, an analytical convolution with this resolutionfunction is of interest. Here, we discuss basic concepts to efficient approaches for fits of QENS spectra basedon standard MATLAB and Python fit algorithms. We illustrate the fits with example data from IN16B, BASIS,and BATS.
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| 4. |
- Beck, Christian, et al.
(author)
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Short-Time Transport Properties of Bidisperse Suspensions of Immunoglobulins and Serum Albumins Consistent with a Colloid Physics Picture.
- 2022
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 126:38, s. 7400-7408
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Journal article (peer-reviewed)abstract
- 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.
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| 5. |
- Beck, Christian, et al.
(author)
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Temperature and salt controlled tuning of protein clusters
- 2021
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In: Soft Matter. - : Royal Society of Chemistry. - 1744-683X .- 1744-6848. ; :37, s. 8506-8516
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Journal article (peer-reviewed)abstract
- 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 (c(p)) and the concentrations of the trivalent salts YCl3 and LaCl3 (c(s)). 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 c(p), c(s) 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(c(p), c(s), 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.
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| 6. |
- Benetti, Ana R, et al.
(author)
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How mobile are protons in the structure of dental glass ionomer cements?
- 2015
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
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Journal article (peer-reviewed)abstract
- The development of dental materials with improved properties and increased longevity can save costs and minimize discomfort for patients. Due to their good biocompatibility, glass ionomer cements are an interesting restorative option. However, these cements have limited mechanical strength to survive in the challenging oral environment. Therefore, a better understanding of the structure and hydration process of these cements can bring the necessary understanding to further developments. Neutrons and X-rays have been used to investigate the highly complex pore structure, as well as to assess the hydrogen mobility within these cements. Our findings suggest that the lower mechanical strength in glass ionomer cements results not only from the presence of pores, but also from the increased hydrogen mobility within the material. The relationship between microstructure, hydrogen mobility and strength brings insights into the material's durability, also demonstrating the need and opening the possibility for further research in these dental cements.
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| 7. |
- Braun, Michal K., et al.
(author)
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Crowding-Controlled Cluster Size in Concentrated Aqueous Protein Solutions : Structure, Self- and Collective Diffusion
- 2017
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In: Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 8:12, s. 2590-2596
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Journal article (peer-reviewed)abstract
- We investigate the concentration-controlled formation of clusters in β-lactoglobulin (BLG) protein solutions combining structural and dynamical scattering techniques. The static structure factor from small-angle X-ray scattering as well as de-Gennes narrowing in the nanosecond diffusion function D(q) from neutron spin echo spectroscopy support a picture of cluster formation. Using neutron backscattering spectroscopy, a monotonous increase of the average hydrodynamic cluster radius is monitored over a broad protein concentration range, corresponding to oligomeric structures of BLG ranging from the native dimers up to roughly four dimers. The results suggest that BLG forms compact clusters that are static on the observation time scale of several nanoseconds. The presented analysis provides a general framework to access the structure and dynamics of macromolecular assemblies in solution.
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| 8. |
- Fagerberg, Eric, et al.
(author)
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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
- 2021
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207.
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Journal article (peer-reviewed)abstract
- 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.
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| 9. |
- Gates, Will P., et al.
(author)
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Neutron Time-of-Flight Quantification of Water Desorption Isotherms of Montmorillonite
- 2012
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In: Journal of Physical Chemistry C. - : American Chemical Society (ACS). - 1932-7447 .- 1932-7455. ; 116:9, s. 5558-5570
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Journal article (peer-reviewed)abstract
- The multiple energy states of water held by surfaces of a clay mineral can be effectively probed with time-of-flight and fixed elastic window neutron scattering. We used these techniques to quantitatively differentiate water types, including rotational and translational diffusions, in Ca- and Na-montmorillonite (SAz-1) and charge-reduced preparations equilibrated at RH = 33% and 55%, whose gravimetric water contents are in proportion with their layer charge. Quasi-elastic neutron scattering results revealed significant differences within interlayer water populations and between interlayer and interparticle waters. Interlayer cationic and H-bonded waters have residence times ranging from a few nanoseconds to tenths of picoseconds, while interparticle water, obtained for the RH = 55% equilibrated samples, showed an average diffusivity faster than interlayer water, yet slower than bulk water. Our results enabled us to differentiate at least two water motions during dehydration of Ca- and Na-SAz-1 (initially equilibrated at RH = 55%) by using a "controlled water loss" time-of-flight procedure. This work confirms that (a) interlayer and cationic water in dioctahedral smectites are characterized by slower motions than interparticle water, (b) interlayer cations influenced the dynamics of water loss, probably through its affect on clay fabric, and (c) interparticle water behaves more like bulk water. At 55% RH the Ca montmorillonite held more interparticle water, but on dehydration under controlled conditions, it retained interlayer and cationic water more strongly than Na montmorillonite.
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| 10. |
- Girelli, Anita, et al.
(author)
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Molecular Flexibility of Antibodies Preserved Even in the Dense Phase after Macroscopic Phase Separation
- 2021
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In: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 18:11, s. 4162-4169
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Journal article (peer-reviewed)abstract
- 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.
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