Spectroscopy of proteins on surfaces: implications for protein orientation and protein-protein interactions

Several approaches for stabilizing three hemeproteins (cytochrome c myoglobin and cytochrome c3) on a silver surface were developed. Methodology for preventing denaturation of complex biomolecules such as surface modification by small molecules is often required in order to obtain surface-enhanced resonance Raman scattering (SERRS) spectra that are relevant to the functional molecule. The optimal approach depends upon the stability of the protein. For example cytochrome is native on a number of different substrates (silver electrodes sols and thin-films) whereas myoglobin is most stable on silver sols. Because SERRS detects changes in the interactions of the heme group with the protein the greater stability of cytochrome as compared to myoglobin probably reflects the fact that the heme is covalently bound in the former. Surface selection rules were used to analyze the orientation of the heme group of several proteins relative to the silver surface. A comparison of the changes in the relative intensities of bands in the SERRS spectra of cytochrome indicates that the protein orientation is sensitive to the presence of lipids or other proteins (avidin) on the silver surface. 1.