Molecular orientation in a hydrated monolayer film of yeast cytochrome c, immobilized via disulfide bonding between Cys-102 and a pyridyl disulfide- capped phospholipid bilayer deposited from an air-water interface onto glass substrates, was investigated. The orientation distribution of the heme groups in the protein film was determined using a combination of absorption linear dichroism, measured in a planarintegrated optical waveguide-attenuated total reflection geometry- and fluorescence anisotropy, measured in a total internal reflection geometry. A gaussian model for the orientation distribution was used to recover the mean heme tilt angle and angular distribution about the mean which were 40 and 11°, respectively. Additional experiments showed that a large fraction of the cytochrome c was disulfide bonded to the bilayer, which correlates with the high degree of macroscopic order in the protein film. However, a subpopulation of yeast cytochrome c molecules in the film (~30% of the total) appeared to be nonspecifically adsorbed. The orientation distribution of this subpopulation was found to be much broader than the specifically bound fraction.
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