Scanning tunneling microscopy (STM) has been used to observe lysozyme at a graphite surface directly in order to gain mechanistic information about the molecular events involved in protein adsorption. The experiments were performed using an insulated tip in an aqueous protein solution, allowing the time course of the adsorption process to be followed, including the evolution of ordered arrays. Ordered arrays of protein molecules were observed, with lattice spacings that varied with bulk protein concentration and salt strength. Fourier analysis was used to determine the average cell dimensions of an array. From the observed lattice spacings, it was possible to estimate the surface coverage of the protein, and thus, by varying the conditions, adsorption isotherms could be obtained. These isotherms compare well with adsorption isotherms measured using total internal reflectance fluorescence (TIRF) spectroscopy on a hydrophobic surface. Since the protein is charged and the electrolyte has an effect on the isotherms, electrostatics are a likely controlling factor. Molecular electrostatics computations were thus used to investigate the possible origins of the lattice structure, and they suggest that favorable intermolecular interactions among adsorbed molecules are consistent with hydrophobically dominated protein-surface interactions.
Two globular proteins, lysozyme and chymotrypsinogen A, were imaged on graphite using a scanning tunneling microscope. In contrast to the isolated molecules typically seen, long-range order was observed in both of these systems when the protein concentration of the solution deposited on the graphite was sufficient for multilayer coverage. For lysozyme, regular two-dimensional arrays of protein molecules were seen, with periodicities ranging from about 40 (the approximate size of a lysozyme molecule) to about 150 Å. These length scales depend on the lysozyme concentration in the initial solution. For chymotrypsinogen A, two-dimensional structures that covered a much smaller area of the graphite were observed. The structural features observed suggest such possibilities as protein structure determination using surface structural techniques and epitaxial growth of protein crystals on these two-dimensional structures.
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