MOLECULAR-ORIENTATION DISTRIBUTIONS IN PROTEIN FILMS .2. SITE-DIRECTED IMMOBILIZATION OF YEAST CYTOCHROME-C ON THIOL-CAPPED, SELF-ASSEMBLEDMONOLAYERS

Molecular orientation in films of yeast cytochrome c immobilized via d isulfide bonding between cysteine 102 and the thiol tail groups of sel f-assembled monolayers (SAMs) coated on planar glass substrates was in vestigated. The orientation distribution of the heme groups in the pro tein film was determined using a combination of absorption linear dich roism, measured in a planar integrated optical waveguide-attenuated to tal reflection geometry, and emission anisotropy, measured in a total internal reflection fluorescence geometry. The mean heme tilt angle an d angular distribution about the mean were recovered using a Gaussian model for the orientation distribution. These data are the first orien tation distribution measurements reported for a protein film immobiliz ed using a site-directed bonding strategy. The results show that the m olecular architecture examined in this study does not produce a highly oriented protein film. A significant fraction of the immobilized cyto chrome c is nonspecifically adsorbed to the SAM surface, which produce s a relatively broad distribution of heme orientations.