Use of X-ray photoelectron spectroscopy in the measurement of protein orientation on surfaces

Abstract

The use of X-ray photoelectron spectroscopy (XPS) for determination of the orientation of proteins on surfaces is demonstrated. A myoglobin derivative in which a pentaamineruthenium(III) group is attached to a histidine residue is used for this purpose, with the ruthenium and the heme iron atoms acting as a double marker. The relative intensities of the X-ray photoelectron spectra for these two markers are shown to be a sensitive measure of orientation in the protein film, since the RuFe distance is comparable to the photoelectron attenuation length. On aluminum, indium-tin oxide and oriented graphite surfaces, XPS indicates that the RuFe axis of the myoglobin derivative is preferentially oriented with the ruthenium furthest from the substrate.