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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 ...

Universität Tübingen

Institut Max von Laue - Paul Langevin (ILL)

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 (DO) 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.

Nanosecond Tracer Diffusion as a Probe of the Solution Structure and Molecular Mobility of Protein Assemblies: The Case of Ovalbumin.

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 (), the protein concentration () and the concentrations of the trivalent salts YCl and LaCl (). As an important complement to structural,  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 ,  and . We determine the cluster size in terms of effective hydrodynamic radii as manifested by the cluster center-of-mass diffusion coefficients . For both salts, we find a simple functional form (, , ) 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.

Temperature and salt controlled tuning of protein clusters.

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  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.

Christian Beck

Institut für Angewandte Physik

Universität Tübingen

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Christian Beck

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Universität Tübingen

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Institut für Angewandte Physik