Imaging the molecular dimensions and oligomerization of protein molecules at the solid-liquid interface by surface oriented molecular sizing (SOMS) microscopy

The structure and behavior of proteins at the solid/liquid interface is of great scientific interest. It has application both to fundamental biochemical understanding, as well as to biotechnological purposes. Interfaces play a critical role in many physiological processes. The mechanism of protein adsorption to surfaces is not very well understood. The current model put forth in much of the literature assumes a two step model. In the first step of this model the protein collides with the surface and adsorbs if its energy is sufficient to overcome the free energy of desorption of surface adsorbed solvent. The second step is often assumed to involve significant conformational change of the secondary and tertiary structure of the protein or enzyme, akin to denaturation. This unfolding of the protein would tend to indicate that loss of function would occur concomitantly, but studies have found very little loss in activity upon adsorption for a number of different protein systems.; The recent development of the atomic force microscope (AFM) offers another tool for the examination of protein structure at liquid/solid interfaces. For atomically flat crystals the AFM has been used to determine atomic positions to {dollar}<{dollar}1 A resolution. In the case of samples with topographic features larger than atoms, the probe tip of the AFM 'convolutes' with the size and shape of surface features. This has hindered the use of AFM for molecular level structural determination of proteins at the liquid/solid interface.; The work presented in this dissertation covers the development of the surface oriented molecular sizing (SOMS) technique which makes use of the angstrom height resolution of the AFM and a physically based mathematical framework for the analysis of the height distribution of adsorbed protein molecules. The surface adsorption and orientation (SAO) model is developed using statistical thermodynamics to model the expected height distributions for molecules adsorbed on a surface.; The SOMS technique will be shown to be viable through studies of ferritin and concanavalin A (Con A) at the water/mica interface. Using this technique we are able to determine both the three-dimensional size and the oligomerization state of the adsorbed molecules. This technique will then be utilized for the examination of denaturation of Con A at the interface, by a number of mechanisms. Further, the structural and orientational changes in Con A as a function of pH will also be presented. The final chapter of this dissertation will present an extension of these studies to the deposition and structure of Con A thin films on mica.