| 1. |
- Beck, Christian, et al.
(författare)
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Following Protein Dynamics in Real Time during Crystallization
- 2019
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Ingår i: Crystal Growth and Design. - : American Chemical Society (ACS). - 1528-7483 .- 1528-7505. ; , s. 7036-7045
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Nanosecond Tracer Diffusion as a Probe of the Solution Structure and Molecular Mobility of Protein Assemblies: The Case of Ovalbumin
- 2018
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Ingår i: The Journal of Physical Chemistry Part B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 122:35, s. 8343-8343
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Tidskriftsartikel (refereegranskat)abstract
- Protein diffusion is not only an important process ensuring biological function but can also be used as a probe to obtain information on structural properties of protein assemblies in liquid solutions. Here, we explore the oligomerization state of ovalbumin at high protein concentrations by means of its short-time self-diffusion. We employ high-resolution incoherent quasielastic neutron scattering to access the self-diffusion on nanosecond timescales, on which interparticle contacts are not altered. Our results indicate that ovalbumin in aqueous (D2O) solutions occurs in increasingly large assemblies of its monomeric subunits with rising protein concentration. It changes from nearly monomeric toward dimeric and ultimately larger than tetrameric complexes. Simultaneously, we access information on the internal molecular mobility of ovalbumin on the nanometer length scale and compare it with results obtained for bovine serum albumin, immunoglobulin, and β-lactoglobulin.
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| 3. |
- Beck, Christian, et al.
(författare)
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Neutron spectroscopy on protein solutions employing backscattering with an increased energy range
- 2019
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Ingår i: Physica B: Condensed Matter. - : Elsevier BV. - 0921-4526. ; 562, s. 31-35
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Tidskriftsartikel (refereegranskat)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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| 4. |
- Beck, Christian, et al.
(författare)
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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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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 126:38, s. 7400-7408
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Temperature and salt controlled tuning of protein clusters
- 2021
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Ingår i: Soft Matter. - : Royal Society of Chemistry. - 1744-683X .- 1744-6848. ; :37, s. 8506-8516
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Tidskriftsartikel (refereegranskat)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. |
- Braun, Michal K., et al.
(författare)
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Crowding-Controlled Cluster Size in Concentrated Aqueous Protein Solutions : Structure, Self- and Collective Diffusion
- 2017
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Ingår i: Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 8:12, s. 2590-2596
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Tidskriftsartikel (refereegranskat)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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| 7. |
- Grimaldo, Marco, et al.
(författare)
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Dynamics of proteins in solution
- 2019
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Ingår i: Quarterly Reviews of Biophysics. - 1469-8994. ; 52
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Forskningsöversikt (refereegranskat)abstract
- The dynamics of proteins in solution includes a variety of processes, such as backbone and side-chain fluctuations, interdomain motions, as well as global rotational and translational (i.e. center of mass) diffusion. Since protein dynamics is related to protein function and essential transport processes, a detailed mechanistic understanding and monitoring of protein dynamics in solution is highly desirable. The hierarchical character of protein dynamics requires experimental tools addressing a broad range of time- and length scales. We discuss how different techniques contribute to a comprehensive picture of protein dynamics, and focus in particular on results from neutron spectroscopy. We outline the underlying principles and review available instrumentation as well as related analysis frameworks.
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| 8. |
- Grimaldo, Marco, et al.
(författare)
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Protein Short-Time Diffusion in a Naturally Crowded Environment
- 2019
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Ingår i: Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 10:8, s. 1709-1715
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Tidskriftsartikel (refereegranskat)abstract
- The interior of living cells is a dense and polydisperse suspension of macromolecules. Such a complex system challenges an understanding in terms of colloidal suspensions. As a fundamental test we employ neutron spectroscopy to measure the diffusion of tracer proteins (immunoglobulins) in a cell-like environment (cell lysate) with explicit control over crowding conditions. In combination with Stokesian dynamics simulation, we address protein diffusion on nanosecond time scales where hydrodynamic interactions dominate over negligible protein collisions. We successfully link the experimental results on these complex, flexible molecules with coarse-grained simulations providing a consistent understanding by colloid theories. Both experiments and simulations show that tracers in polydisperse solutions close to the effective particle radius R eff = R i 3 1/3 diffuse approximately as if the suspension was monodisperse. The simulations further show that macromolecules of sizes R > R eff (R < R eff ) are slowed more (less) effectively even at nanosecond time scales, which is highly relevant for a quantitative understanding of cellular processes.
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| 9. |
- Lenton, Samuel, et al.
(författare)
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Effect of Phosphorylation on a Human-like Osteopontin Peptide
- 2017
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495. ; 112:8, s. 1586-1596
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Tidskriftsartikel (refereegranskat)abstract
- The last decade established that the dynamic properties of the phosphoproteome are central to function and its modulation. The temporal dimension of phosphorylation effects remains nonetheless poorly understood, particularly for intrinsically disordered proteins. Osteopontin, selected for this study due to its key role in biomineralization, is expressed in many species and tissues to play a range of distinct roles. A notable property of highly phosphorylated isoforms of osteopontin is their ability to sequester nanoclusters of calcium phosphate to form a core-shell structure, in a fluid that is supersaturated but stable. In Biology, this process enables soft and hard tissues to coexist in the same organism with relative ease. Here, we extend our understanding of the effect of phosphorylation on a disordered protein, the recombinant human-like osteopontin rOPN. The solution structures of the phosphorylated and unphosphorylated rOPN were investigated by small-angle x-ray scattering and no significant changes were detected on the radius of gyration or maximum interatomic distance. The picosecond-to-nanosecond dynamics of the hydrated powders of the two rOPN forms were further compared by elastic and quasi-elastic incoherent neutron scattering. Phosphorylation was found to block some nanosecond side-chain motions while increasing the flexibility of other side chains on the faster timescale. Phosphorylation can thus selectively change the dynamic behavior of even a highly disordered protein such as osteopontin. Through such an effect on rOPN, phosphorylation can direct allosteric mechanisms, interactions with substrates, cofactors and, in this case, amorphous or crystalline biominerals.
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| 10. |
- Matsarskaia, Olga, et al.
(författare)
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Evolution of the structure and dynamics of bovine serum albumin induced by thermal denaturation
- 2020
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry. - 1463-9076 .- 1463-9084. ; 22, s. 18507-18517
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Tidskriftsartikel (refereegranskat)abstract
- Protein denaturation in concentrated solutions consists of the unfolding of the native protein structure, and subsequent cross-linking into clusters or gel networks. While the kinetic evolution of structure has been studied for some cases, the underlying microscopic dynamics of proteins has so far been neglected. However, protein dynamics is essential to understand the specific nature of assembly processes, such as diffusion-limited growth, or vitrification of dense liquids. Here, we present a study on thermal denaturation of concentrated solutions of bovine serum albumin (BSA) in D2O with and without NaCl. Using small-angle scattering, we provide information on structure before, during and after denaturation. Using quasi-elastic neutron scattering, we monitor in real-time the microscopic dynamics and dynamical confinement throughout the entire denaturation process covering protein unfolding and cross-linking. After denaturation, the protein dynamics is slowed down in salty solutions compared to those in pure water, while the stability and dynamics of the native solution appears unaffected by salt. The approach presented here opens opportunities to link microscopic dynamics to emerging structural properties, with implications for assembly processes in soft and biological matter.
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| 11. |
- Sohmen, Benedikt, et al.
(författare)
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The Onset of Molecule-Spanning Dynamics in Heat Shock Protein Hsp90
- 2023
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Ingår i: Advanced Science. - : John Wiley & Sons. - 2198-3844. ; 10:36
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 12. |
- Squazzoni, Flaminio, et al.
(författare)
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Unlock ways to share data on peer review
- 2020
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Ingår i: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 578:7796, s. 512-514
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Tidskriftsartikel (refereegranskat)
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