Two-dimensional crystallization of streptavidin studied by quantitative Brewster angle microscopy

Brewster angle microscopy (BAM) is extended to probe protein adsorption and aggregation processes at interfaces quantitatively. Since no p-polarized light is reflected from the plain air-water interface under Brewster's angle, the grayscales of BAM images solely depend on the optical thicknesses of interfacial layers; photolabeling is not required. Effective refractive indices are calculated from the grayscales via Fresnel's equations and are finally converted to relative protein surface densities by the Maxwell-Garnett theory. It is shown here that the Maxwell-Garnett theory provides an accurate framework for the description of a protein-water slab. Streptavidin binding to biotinylated monolayers at the air-water interface was chosen as a model system for an in situ study of the formation of two-dimensional streptavidin crystals. H-shaped aggregates are seen with BAM. We identify these as two-dimensional crystals since they are equal in size and shape to those typically observed in fluorescence microscopy which have been characterized by electron diffraction. The difference in protein surface density between the streptavidin crystals and the noncrystalline surrounding is sufficient to provide for a rich contrast without the use of a fluorescence probe. A critical protein surface density equal to 75% of the crystal density is found to be required for the crystals to form, a value that is independent of the protein bulk concentration. We have studied further the compressibility of the two phases. Whereas the density of the crystalline phase remains constant during compression, the noncrystalline phase can be compressed to a surface density which exceeds that of the crystalline phase without initiating further crystal growth. This leads to an inverted contrast in BAM; dark crystals are seen on a bright background. The protein density of the noncrystalline phase can also exceed that of the crystalline phase upon the much slower process of protein adsorption from sufficiently concentrated bulk solutions. The nature of this two-dimensional phase transition is discussed.