MOLECULAR-ORIENTATION DISTRIBUTIONS IN PROTEIN FILMS - 4 - A MULTILAYER COMPOSED OF YEAST CYTOCHROME-C BOUND THROUGH AN INTERMEDIATE STREPTAVIDIN LAYER TO A PLANAR SUPPORTED PHOSPHOLIPID-BILAYER

Multilayer protein films in which avidin or streptavidin is used as a cross-linking agent are widely used in bioanalytical laboratories for diagnostics, isolation, and localization. However, comparatively littl e is known about the structure of these films. In this study, molecula r orientation in asymmetric protein bilayers assembled on planar glass substrates was investigated. The multilayer architecture consisted of , in order of deposition, a biotin capped phospholipid bilayer, strept avidin, and yeast cytochrome c biotinylated at cysteine 102. The orien tation distribution of the heme groups in the cytochrome c layer was d etermined using a combination of absorption linear dichroism, measured in a planar integrated optical waveguide-attenuated total reflection geometry, and fluorescence anisotropy, measured in a total internal re flection geometry. A Gaussian model for the orientation distribution w as used to recover the mean heme tilt angle and angular distribution a bout the mean. The orientation distribution for the biotinylated cytoc hrome c layer was 41 +/- 11 degrees, which is nearly identical to the orientation distribution measured previously for yeast cytochrome c co valently immobilized via cysteine 102 to pyridyl-capped phospholipid b ilayers.(12) In that case, the cytochrome surface coverage was 0.9 mon olayer. In the present study the cytochrome surface coverage was only 0.4 monolayer, which likely precluded lateral interactions between pro tein molecules. The fact that the orientation distributions were nearl y identical shows that lateral interactions are not required to form a macroscopically oriented protein film.