Proteins in the intracellular environment occur in highly concentrated "crowded" aqueous solutions
of different macromolecules and salts. Both the molecular crowding and the presence of different salts affect
the mobility and in particular the diffusion of the proteins. Salts also affect protein aggregation and induce
complex phenomena such as the reentrant condensation, i.e. a solubility which is not monotonously related to the
ionic strength. We investigate these issues on simplified model systems by applying neutron spectroscopy and
complementary techniques to study globular proteins and salts with different valency in aqueous solution.
Proteins in solution form monodisperse
colloidal suspensions. In addition to their biological role, proteins in solution are therefore of fundamental
interest in the context of soft matter science. Proteins, however, differ in one important aspect from many
simple colloidal systems: The distribution of charges on the surface of a protein is in general
inhomogeneous. This inhomogeneous surface charge distribution can in turn be assumed to have a fundamental
biological relevance in controlling for instance aggregation phenomena and biological activity such as docking
processes. Characteristic of proteins in their native environment is the macromolecular crowding -
i.e. relatively large volume fractions being occupied by proteins - and the aqueous solvent containing salt
ions. These salt ions are crucial for the understanding of the effective interactions of proteins and the
resulting structures as well as indeed the dynamics. |
For this project we apply quasi-elastic neutron scattering using for instance the cold neutron backscattering spectrometer IN16B and the neutron spin-echo spectrometers IN15 and WASP within the Spectroscopy Group at the ILL. In addition, we employ small angle X-ray and neutron scattering, dynamic light scattering and other complementary techniques. Here, we study static and dynamic aspects of proteins in aqueous solutions containing salts. These address the issues of crowding and of the effects of monovalent salts for the model protein Bovine Serum Albumin (BSA) [3], using a combination of small-angle X-ray scattering (SAXS), and the quasi-elastic neutron scattering (QENS) techniques backscattering (BS), and spin-echo (NSE). SAXS thereby accesses the static structure on protein-protein nearest neighbour distances, whereas BS and NSE provide information on different regimes of the protein diffusion on nanosecond time scales. From SAXS data we find a qualitative change with rising protein concentration from an uncorrelated to a strongly correlated solution. For weaker charge screening (i.e. less salt) this correlation is found already for lower protein concentrations. We conclude that below a volume fraction of approximately 10% crowding is induced by unscreened charges. In this case the SAXS correlation peak disappears by the addition of NaCl due to the salt screening effect (Fig. 2), i.e. with increasing ionic strength the surface potential decays faster with distance and reduces the long-ranged repulsion between protein molecules. By contrast, above that volume fraction crowding is dominated by the excluded-volume contribution, and the SAXS correlation peak is conserved upon salt addition (data not shown). The conservation of the correlation peak also for higher salt concentrations implies the absence of aggregation in this highly concentrated protein solution.
Neutron backscattering and spin-echo probe different regimes of diffusion due to
the different scattering vector ranges accessed by these techniques and different sensitivity to coherent and
incoherent scattering. The scattering vectors accessed by spin-echo are approximately commensurate with those
accessed by SAXS, whilst backscattering measures at larger vectors corresponding to intramolecular length
scales. From the backscattering and spin-echo data we find a continuously changing behaviour of the
self-diffusion of the proteins due to the excluded-volume effect. The addition of salt has little or no effect
on the apparent diffusion coefficients observed in backscattering (Fig. 3), although charge screening is assumed
to change both interaction time and coupling strength. In contrast to backscattering data, we see an increase of
diffusion upon addition of salt in neutron spin echo data (Fig. 4), whereas the dependence on protein
concentration remains qualitatively the same, i.e., a decrease of apparent diffusion upon increasing protein
concentration. In the protein concentration range thus far covered by our experiments, i.e. from approximately
4% to 27% volume fraction, our data are in agreement with a continuous decrease of the apparent diffusion
constants with the protein concentration. In contrast to the static data, our dynamic data show no distinct
value where crowding due to the excluded-volume contribution sets in. |
[1] F. Zhang, M. Skoda, R. Jacobs, et al.,
Phys. Rev. Lett. 101 (2008) 14 .
[2] A. Y.Grosberg, T. T. Nguyen, B. I. Shklovskii, Rev. Modern
Phys. 74 (2002) 329.
[3] F. Roosen-Runge, M. Hennig, T. Seydel, F. Zhang, M. W.A. Skoda,
S. Zorn, R. Jacobs, M. Maccarini, P. Fouquet,
F. Schreiber; BBA - Proteins and Proteomics 1804 (2010) 68.