Imaging of protein adsorption and self-assembled proteins at the solid-liquid interface by atomic force microscopy

Protein adsorption and self-assembly at the solid/liquid interface occur in a variety of different situations including medical, analytical, biotechnical, industrial, and personal applications. To control adsorption at the interface, it is important to understand the fundamentals behind this process. To this end, atomic force microscopy (AFM) is used to obtain real-time, in situ, spatial distribution information of protein adsorption and protein self-assembly at the solid/liquid interface. The AFM provides lateral distribution data that other techniques traditionally used to study protein adsorption cannot obtain. Thus, AFM data of protein adsorption complements data from these other techniques to provide a better understanding of these processes.;The adsorption dynamics of hen-egg-white lysozyme (HEWL) onto muscovite mica are followed using in situ atomic force microscopy (AFM) in aqueous solution. Under stagnant conditions at a low bulk concentration of protein (2 mug/mL), clusters of protein were readily visible on the mica surface after a few minutes of exposure to the protein solution. Force-volume imaging confirmed the presence of single-layer lysozyme aggregates surrounded by mica. The mass of the protein clusters was measured by using 14 C-labeled lysozyme. The radiolabeled protein experiments in conjunction with AFM images show that the clusters formed at early times average about five protein monomers in size. At higher bulk protein concentration (5 mug/mL), a complete monolayer gradually forms, proceeded by the formation of a second layer of lysozyme. Washout experiments performed on the submonolayer and multilayer adsorption conditions for both early and late times indicate that the protein is irreversibly adsorbed with respect to dilution. The AFM data suggests a model by which the protein irreversibly adsorbs onto the solid surface and proceeds to diffuse on the surface. Upon adsorption, the conformation of the protein changes, exposing previously buried hydrophobic residues that, upon surface collision, can interact with hydrophobic residues of neighboring proteins to form aggregates.;AFM is used to study the self-assembly of bacteriorhodopsin (bR) within reconstituted purple membrane (RPM) adsorbed onto mica. The five RPMs include: RPM 3 (bR/native polar lipids/dimyristoylphosphatidylcholine (DMPC) in a 1:9:14 molar ratio), RPM 4 (bR/native polar lipids in a 1:7 molar ratio), RPM 5 (bR/native polar lipids/1,2-di-O-phytanyl-sn-glycerol in a 1:3.5:6.1 molar ratio), RPM 6 (bR/native polar lipids/1,2-di- O-phytanyl-sn-glycero-3-phosphocholine in a 1:3.5:4.9 molar ratio), and RPM 7 (bR/native polar lipids/1,2-diphytanoyl-sn -glycero-3-[phospho-L-serine] in a 1:3.5:4.6 molar ratio). (Abstract shortened by UMI.)...