New Methods to Study Biomolecules at Liquid Surfaces
Mark L. Schlossman
University of Illinois at Chicago, Departments of Physics and Chemistry,
845 W. Taylor St., Chicago, IL 60607, schloss@uic.edu
We discuss two new methods to study biomolecules, specifically proteins, at liquid surfaces. The first consists of the insertion of proteins into thin aqueous films supported on aqueous subphases. This involves new methods to make these films and study them with x-rays. The second is a new method of analysis of x-ray reflectivity from proteins adsorbed to lipid monolayers supported on an aqueous buffer subphase.
Aqueous Films: The formation of thin, nanoscale, aqueous films on top of an aqueous subphase will be discussed. Aqueous biphase solutions of poly(ethylene glycol), potassium phosphates, and water are used to form these films. The films form through a spreading process that results in the coexistence of macroscopic lenses and films that are several nanometers thick. Synchrotron x-ray scattering is used to characterize the structure of the thin film and to probe the adsorption of proteins to the film.
X-ray reflectivity measures the layer thickness (4 to 5 nm) and the roughness of the two interfaces of the film. Surface and interfacial tension measurements, combined with the reflectivity measurements, indicate that the films are a thin layer of the bulk solution rather than a monolayer of PEG molecules. X-ray off-specular diffuse scattering determines a coupling constant that characterizes the coupled capillary wave fluctuations of the liquid-vapor and liquid-liquid interfaces of this thin film. Combining these measurements allows the film to be described by an excess free energy with a short range piece (due primarily to capillary wave entropic repulsion) and a long range van der Waals interaction.
Biomolecules, such as proteins, can be trapped at the aqueous-aqueous interface or in the thin film. This idea is demonstrated by an x-ray reflectivity study of ferritin proteins that form a 2-dimensional array at the interface. It is shown that the electron density interfacial profile of the ferritin trapped in this thin film is consistent with the known crystal structure of ferritin. In the absence of the thin film, ferritin does not adsorb to the interface.
Proteins Adsorbed to Lipid Monolayers: X-ray reflectivity from peripheral membrane proteins adsorbed to Langmuir monolayers of lipids has been used many times to probe the lipid-protein interaction. Although this technique does not have the atomic resolution of x-ray crystallography, it is able to probe the structure of a disordered system of proteins bound to a layer of lipids. We demonstrate that, in certain cases, the high resolution crystal structure of the proteins can be usefully incorporated into the analysis of x-ray reflectivity data to yield more information than is normally available from a slab-model analysis of these data. This analysis method is applied to the interaction of C2 domains of cytosolic phospholipase (cPLA 2-C2) with a Langmuir monolayer of SOPC (1-steraoyl-2-oleoyl-sn-glycero-3-phosphocholine) supported on a buffered aqueous solution containing Ca 2+. The analysis determines the angular orientation and penetration depth of the cPLA 2-C2 domains into the SOPC monolayer. We also demonstrate the conditions necessary to measure x-ray reflectivity from Langmuir monolayers of unsaturated lipids, such as SOPC, with negligible x-ray damage.
