This article describes the use of modern light scattering analytical methods to ensure that protein samples about to be analysed by small angle neutron scattering (SANS) techniques are not aggregated, which would otherwise result in poor quality of SANS data. The original studies were carried out by Dr J. Taylor of the University of Portsmouth, UK, in his work on the multimeric restriction endonuclease EcoR1241.
Whereas light usually just scatters off the surface of an object to reveal a visual image, X-rays and neutrons have enough energy to penetrate the structure and scatter off the internal arrays of atoms. The scattering process for neutrons and X-rays is different, however. X-rays are scattered by the electrons in atoms whereas neutrons are scattered by the atomic nuclei. The wavelength of neutrons makes them suitable for investigating molecules of sizes ranging from about 1 to 100 nanometres. One variation of the neutron technique which involves scattering neutrons at very small angles (small angle neutron scattering, or SANS) is used to resolve larger, intermolecular distances in complexes of several molecules. SANS analysis can be carried out at the Institut Laue Langevin (ILL) in Grenoble, France. Since the allocation of beam time at ILL is in great demand it is important to ensure that the sample to be tested is in the optimal condition to ensure the highest quality of data. In particular, non-aggregated samples are essential for small-angle neutron scattering (SANS).
In Dr J. Taylor’s work on the multimeric restriction endonuclease molecule EcoR1241, although the protein samples looked fine by SDS-PAGE and UV-spectoscopy (as judged by the flat baseline and low minima at 260 nm), several SANS measurements showed that some of the protein samples were prone to aggregation and hence poor measurements were obtained.
To better monitor the aggregation status of the protein sample prior to SANS analysis, dynamic light scattering analysis was carried out using a DynaPro MSTC800 light scattering instrument and the monodispersity of the protein samples following purification. From thirty successful measurements, values for the hydrodynamic radius, Rh, and polydispersity were obtained using % mass calculations [Figure 1]. The experimental molecular weight, Mr, was calculated using a volume shape hydration model and was compared to the theoretical Mr, and the two values were found to be in very good agreement. The sample was non-aggregated and thus suitable for SANS measurements. Dr Taylor’s group now routinely use dynamic light scattering to monitor the behaviour of the protein in solution. This has enabled them to determine which of the proteins can be stored and which need to be purified “fresh” prior to complex formation. The maximum concentration of the proteins prior to the formation of aggregates has thus been determined and has enabled the University of Portsmouth team to obtain the best quality SANS data, in the shortest measuring time at ILL, Grenoble, France.
Wyatt Technology
Santa Barbara, CA, USA.
