A method for studying protein orientation with atomic force microscopy using relative protein volumes

• A method for studying protein orientation is described, in which the relative volumes of single proteins and single molecule complexes are measured using atomic force microscopy (AFM). Site-specific ligands are used as “probes” to bind to surface adsorbed proteins. The quantity of formed complexes gives an estimate of the amount of protein oriented in such a way as to allow ligand binding. The volume distribution for single proteins adsorbed to a surface was calculated and fitted to a Gaussian function. This volume distribution was used to localize the same proteins on surfaces with protein−ligand complexes, thus rendering it possible to find the amount of complex formed. Two model systems were used: one with two different mouse monoclonal antibodies of IgG1 type (mAb's against human serum transferrin, hST) adsorbed on silicon surfaces, and one with hST adsorbed to unmodified mica and aminated mica. The adsorbed proteins were allowed to react with a site-specific ligand, which binds to a defined region of the adsorbed protein (hST in the case of adsorbed mAb and lectin in the case of adsorbed hST). A great difference in ligand binding was found between the two antibodies adsorbed to the same type of surface as well as between hST adsorbed to different surfaces. This differance can be attributed to different orientation of the proteins on the surface. The general approach of this method suggests that it can be used for almost any site-specific molecule, either for surface orientation studies or studies where single molecule interactions need to be investigated.

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