91 research outputs found
Arrest transitions in protein solutions – insight from combining scattering, microrheology, and computer simulations
The static and dynamic properties of concentrated protein solutions are essential ingredients for our understanding of the cellular machinery or formulating biopharmaceuticals. Here a combination of advanced characterization techniques such as light and x-ray scattering, neutron spin echo measurements [1] and microrheology experiments [2], combined with the theoretical toolbox from colloid physics and state-of-the-art computer simulations [3], considerably enhances our understanding of the link between protein interactions and the stability, dynamics and flow properties of these solutions up to high concentrations. We will address the enormous influence of weak attractive interactions known to exist between many globular proteins, and demonstrate the dramatic effect of an interaction potential anisotropy [1] such as attractive patches and shape anisotropy [3] on the dynamic properties. We will also discuss how we can combine interparticle interaction effects and the formation of (transient) equilibrium clusters in an attempt to understand and predict properties such as the concentration dependence of the zero shear viscosity of dense protein solutions [4].
(1) Bucciarelli, S.; Myung, J. S.; Farago, B.; Das, S., Vliegenthart, G.; Holderer, O.; Winkler, R. G.; Schurtenberger, P.; Gompper, G.; Stradner, A. “Dramatic influence of patchy attractions on short-time protein diffusion under crowded conditions” Sci. Adv. 2016, 2:e1601432.
(2) Garting, T. and Stradner, A. “Optical Microrheology of Protein Solutions using Tailored Nanoparticles” Small 2018, 1801548.
(3) Myung, J. S.; Roosen-Runge, F.; Winkler, R. G.; Gompper, G.; Schurtenberger, P.; Stradner, A. “Weak shape anisotropy leads to non-monotonic crowding effects impacting protein dynamics under physiologically relevant conditions” J. Phys. Chem. B 2018, 122, 12396-12402.
(4) Bergman, M.; Garting, T.; Schurtenberger, P.; Stradner, A. “Experimental Evidence for a Cluster Glass Transition in Concentrated Lysozyme Solutions” submitted to J. Phys. Chem. B, 2019
Modeling Equilibrium Clusters in Lysozyme Solutions
We present a combined experimental and numerical study of the equilibrium
cluster formation in globular protein solutions under no-added salt conditions.
We show that a cluster phase emerges as a result of a competition between a
long-range screened Coulomb repulsion and a short-range attraction. A simple
effective potential, in which only depth and width of the attractive part of
the potential are optimized, accounts in a remarkable way for the wavevector
dependence of the X-ray scattering structure factor.Comment: 4 pages, 4 figure
Interplay between Spinodal Decomposition and Glass Formation in Proteins Exhibiting Short-Range Attractions
We investigate the competition between spinodal decomposition and dynamical
arrest using aqueous solutions of the globular protein lysozyme as a model
system for colloids with short-range attractions. We show that quenches below a
temperature Ta lead to gel formation as a result of a local arrest of the
proteindense phase during spinodal decomposition. The rheological properties of
these gels allow us to use centrifugation experiments to determine the local
densities of both phases and to precisely locate the gel boundary and the
attractive glass line close to and within the unstable region of the phase
diagram
Interplay between spinodal decomposition and glass formation in proteins exhibiting short-range attractions
We investigate the competition between spinodal decomposition and dynamical arrest using aqueous solutions of the globular protein lysozyme as a model system for colloids with short-range attractions. We show that quenches below a temperature Ta lead to gel formation as a result of a local arrest of the protein-dense phase during spinodal decomposition. The rheological properties of these gels allow us to use centrifugation experiments to determine the local densities of both phases and to precisely locate the gel boundary and the attractive glass line close to and within the unstable region of the phase diagram
A colloid approach to self-assembling antibodies
Concentrated solutions of monoclonal antibodies have attracted considerable
attention due to their importance in pharmaceutical formulations, yet their
tendency to aggregate and the resulting high solution viscosity has posed
considerable problems. It remains a very difficult task to understand and
predict the phase behavior and stability of such solutions. Here we present a
systematic study of the concentration dependence of the structural and dynamic
properties of monoclonal antibodies using a combination of different scattering
methods and microrheological experiments. To interpret these data, we use a
colloid-inspired approach based on a simple patchy model, which explicitly
takes into account the anisotropic shape and the charge distribution of the
molecules. Combining theory, simulations and experiments, we are able to
disentangle self-assembly and intermolecular interactions and to quantitatively
describe the concentration dependence of structural and dynamic quantities such
as the osmotic compressibility, the collective diffusion coefficient and the
zero shear viscosity over the entire range of investigated concentrations. This
simple patchy model not only allows us to consistently describe the
thermodynamic and dynamic behavior of mAb solutions, but also provides a robust
estimate of the attraction between their binding sites. It will thus be an
ideal starting point for future work on antibody formulations, as it provides a
quantitative assessment of the effects of additional excipients or chemical
modifications on antibody interactions, and a prediction of their effect on
solution viscosity
Accurate Correction of the "bulk Response" in Surface Plasmon Resonance Sensing Provides New Insights on Interactions Involving Lysozyme and Poly(ethylene glycol)
Surface plasmon resonance is a very well-established surface sensitive technique for label-free analysis of biomolecular interactions, generating thousands of publications each year. An inconvenient effect that complicates interpretation of SPR results is the "bulk response"from molecules in solution, which generate signals without really binding to the surface. Here we present a physical model for determining the bulk response contribution and verify its accuracy. Our method does not require a reference channel or a separate surface region. We show that proper subtraction of the bulk response reveals an interaction between poly(ethylene glycol) brushes and the protein lysozyme at physiological conditions. Importantly, we also show that the bulk response correction method implemented in commercial instruments is not generally accurate. Using our method, the equilibrium affinity between polymer and protein is determined to be KD = 200 ÎĽM. One reason for the weak affinity is that the interaction is relatively short-lived (1/koff < 30 s). Furthermore, we show that the bulk response correction also reveals the dynamics of self-interactions between lysozyme molecules on surfaces. Besides providing new insights on important biomolecular interactions, our method can be widely applied to improve the accuracy of SPR data generated by instruments worldwide
Phase separation and dynamical arrest for particles interacting with mixed potentials--the case of globular proteins revisited
We examine the applicability of the extended law of corresponding states
(ELCS) to equilibrium and non equilibrium features of the state diagram of the
globular protein lysozyme. We provide compelling evidence that the ELCS
correctly reproduces the location of the binodal for different ionic strengths,
but fails in describing the location of the arrest line. We subsequently use
Mode Coupling Theory (MCT) to gain additional insight into the origin of these
observations. We demonstrate that while the critical point and the connected
binodal and spinodal are governed by the integral features of the interaction
potential described by the normalized second virial coefficient, the arrest
line is mainly determined by the attractive well depth or bond strength. This
article is published in Soft Matter. The reference is: DOI: 10.1039/c0sm01175
Using cluster theory to calculate the experimental structure factors of antibody solutions
Monoclonal antibody solutions are set to become a major therapeutic tool in
the years to come, capable of targeting various diseases by clever designing
their antigen binding site. However, the formulation of stable solutions
suitable for patient self-administration typically presents challenges, as a
result of the increase in viscosity that often occurs at high concentrations.
Here, we establish a link between the microscopic molecular details and the
resulting properties of an antibody solution through the characterization of
clusters, which arise in the presence of self-associating antibodies. In
particular, we find that experimental small-angle X-ray scattering data can be
interpreted by means of analytical models previously exploited for the study of
polymeric and colloidal objects, based on the presence of such clusters. The
latter are determined by theoretical calculations and supported by computer
simulations of a coarse-grained minimal model, in which antibodies are treated
as Y-shaped colloidal molecules and attractive domains are designed as patches.
Using the theoretically-predicted cluster size distributions, we are able to
describe the experimental structure factors over a wide range of concentration
and salt conditions. We thus provide microscopic evidence for the
well-established fact that the concentration-dependent increase in viscosity is
originated by the presence of clusters. Our findings bring new insights on the
self-assembly of monoclonal antibodies, which can be exploited for guiding the
formulation of stable and effective antibody solutions
A new instrument for time-resolved static and dynamic light-scattering experiments in turbid media
We present a new 3D cross-correlation instrument that not only allows for static and dynamic scattering experiments with turbid samples but measures at four angles simultaneously. It thus extends the application of cross-correlation light scattering to time-resolved studies where we can, for example, efficiently investigate the temporal evolution of aggregating or phase separating turbid dispersions. The combination of multiangle 3D and on-line transmission measurements is an essential prerequisite for such studies. This not only provides time-resolved information about the overall size and shape of the particles through measurements of the mean apparent radius of gyration and hydrodynamic radius, but also on the weight-average apparent molar mass via the absolute forward scattering intensity. We present an efficient alignment strategy based on the novel design of the instrument and then the application range of the instrument using well-defined model latex suspensions. The effectiveness of the cross-correlation multiangle technique to monitor aggregation processes in turbid suspensions is finally shown for the acidification of skim milk during the yoghurt-making process. Due to the self-assembled nature of the casein micelles an understanding of the sol–gel process induced by the acidification is only feasible if time-resolved light-scattering experiments on an absolute scale are possible under industrially relevant conditions, where the casein solutions are highly turbid
Theoretical predictions of structures in dispersions containing charged colloidal particles and non-adsorbing polymers
We develop a theoretical model to describe structural effects on a specific system of charged colloidal polystyrene particles, upon the addition of non-adsorbing PEG polymers. This system has previously been investigated experimentally, by scattering methods, so we are able to quantitatively compare predicted structure factors with corresponding experimental data. Our aim is to construct a model that is coarse-grained enough to be computationally manageable, yet detailed enough to capture the important physics. To this end, we utilize classical polymer density functional theory, wherein all possible polymer configurations are accounted for, subject to a mean-field Boltzmann weight. We make efforts to counteract drawbacks with this mean-field approach, resulting in structural predictions that agree very well with computationally more demanding simulations. Electrostatic interactions are handled at the fully non-linear Poisson-Boltzmann level, and we demonstrate that a linearization leads to less accurate predictions. The particle charge is an experimentally unknown parameter. We define the surface charge such that the experimental and theoretical gel point at equal polymer concentration coincide. Assuming a fixed surface charge for a certain salt concentration, we find very good agreements between measured and predicted structure factors across a wide range of polymer concentrations. We also present predictions for other structural quantities, such as radial distribution functions, and cluster size distributions. Finally, we demonstrate that our model predicts the occurrence of equilibrium clusters at high polymer concentrations, but low particle volume fractions and salt levels
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