MOLECULAR-ORIENTATION DISTRIBUTIONS IN PROTEIN FILMS - III - YEAST CYTOCHROME-C IMMOBILIZED ON PYRIDYL-DISULFIDE-CAPPED PHOSPHOLIPID-BILAYERS

Molecular orientation in a hydrated monolayer film of yeast cytochrome c, immobilized via disulfide bonding between Cys-102 and a pyridyl di sulfide-capped phospholipid bilayer deposited from an air-water interf ace onto glass substrates, was investigated. The orientation distribut ion of the heme groups in the protein film was determined using a comb ination of absorption linear dichroism, measured in a planarintegrated optical waveguide-attenuated total reflection geometry-and fluorescen ce anisotropy, measured in a total internal reflection geometry. A gau ssian model for the orientation distribution was used to recover the m ean heme tilt angle and angular distribution about the mean, which wer e 40 and 11 degrees, respectively. Additional experiments showed that a large fraction of the cytochrome c was disulfide bonded to the bilay er, which correlates with the high degree of macroscopic order in the protein film. However, a subpopulation of yeast cytochrome c molecules in the film (similar to 30% of the total) appeared to be nonspecifica lly adsorbed. The orientation distribution of this subpopulation was f ound to be much broader than the specifically bound fraction.