SANS Observation of Bacteriorhodopsin Precrystallization Intermediates in the Lipidic Cubic Phase

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Friday, November 3, 2017 - 11:00am to 12:00pm
Thomas Cleveland
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Membrane proteins can be incorporated into the lipidic cubic phase (LCP) for crystal growth and structure determination. LCP crystallization has become an important tool in the field of membrane protein crystallography (particularly, but not solely, with GPCRs and other small membrane proteins). However, many details of this process are not well understood. There is little direct experimental evidence for the localization of protein and detergent after incorporation into LCP; the mechanisms of nucleation and crystal growth; and the details of how the cubic phase modifies the interactions between protein molecules. We are using Small Angle Neutron and X-Ray Scattering (SANS/SAXS) to study each step of the cubic phase crystallization process using Bacteriorhodopsin (bR) as a model system.

Using SANS, it is possible to contrast-match the non-protein components of the system, i.e. detergents and lipids. This allows us to measure the protein scattering directly and in isolation, greatly simplifying the data interpretation from these complex multicomponent systems. At high bR concentrations, it is possible to measure structure factors, from which information on protein-protein interactions can be obtained. We have measured the concentration-dependent scattering of bR: (1) in solution; (2) after incorporation into LCP; and (3) as a function of precipitant concentration. Solution structure factor measurements at lower salt concentrations are consistent with a charged sphere interaction model. In contrast-matched LCP at lower concentrations of bR and precipitant, scattering from bR monomers could be observed, similarly to bR in solution. At higher bR and precipitant concentrations, a series of higher-order structures were observed by SANS, as well as protein-dependent Bragg reflections in samples in which macroscopic crystals were later observed.

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