The formation of chemisorbed monolayers of yeast cytochrome c on both uncharged polar and nonpolar soft surfaces of organic self-assembled monolayers (SAM) on solid inorganic substrates was followed in situ by polarized total internal reflection fluorescence. Two types of nonpolar surfaces and one type of uncharged polar surface were used. The first type of nonpolar surface contained only thiol endgroups, while the other was composed of a mixture of thiol and methyl endgroups. The uncharged polar surface was provided by the mixture of thiol and hydroxyl endgroups. The thiol endgroups were used to form a covalent disulfide bond with the unique surface-exposed cysteine residue 102 of the protein. The mean tilt angle of the protein's zinc-substituted porphyrin was found to be 41 degrees and 50 degrees for the adsorption onto the nonpolar and uncharged polar surfaces, respectively. The distribution widths for the pure thiol and the thiol/methyl and thiol/hydroxyl mixtures were 9 degrees, 1 degrees, and 18 degrees, respectively. The high degree of the orientational order and good stability achieved for the protein monolayer on the mixed thiol/methyl endgroup SAM makes this system very attractive for studies of both intramolecular and intermolecular electron transfer processes.

Polarized x-ray absorption fine structure (XAFS) spectroscopy has been performed in fluorescence mode under total external reflection conditions on frozen hydrated single monolayers of yeast cytochrome c (YCC). The protein molecules were vectorially oriented within the monolayer by tethering their naturally occurring and unique surface cysteine residues to the sulfhydryl-endgroups at the surface of a mixed organic self-assembled monolayer, itself covalently attached to an ultrapure silicon wafer. The sulfhydryl-endgroups were isolated by dilution with either methyl- or hydroxyl-endgroups, producing macroscopically nonpolar or uncharged-polar soft surfaces, respectively. Independent information on the heme-plane orientation relative to the monolayer plane was obtained experimentally via optical linear dichroism. The polarized XAFS data have been analyzed both qualitatively and by a global mapping approach limited to systematically altering the various iron-ligand distances within a model for the local atomic environment of the heme prosthetic group, and comparing the theoretically generated XAFS spectra with those obtained experimentally. A similar analysis of unpolarized XAFS data from a frozen solution of YCC was performed using either the heme environment from the NMR solution or the x-ray crystallographic data for YCC as the model structure. All resulting iron-ligand distances were then used in molecular dynamics (MD) computer simulations of YCC in these three systems to investigate the possible effects of anisotropic ligand motions on the fits of the calculated to the experimental XAFS spectra.

Yeast cytochrome c (YCC) can be covalently tethered to, and thereby vectorially oriented on, the soft surface of a mixed endgroup (e.g., -CH3/-SH = 6:1, or -OH/-SH = 6:1) organic self-assembled monolayer (SAM) chemisorbed on the surface of a silicon substrate utilizing a disulfide linkage between its unique surface cysteine residue and a thiol endgroup. Neutron reflectivities from such monolayers of YCC on Fe/Si or Fe/Au/Si multilayer substrates with H2O versus D2O hydrating the protein monolayer at 88% relative humidity for the nonpolar SAM (-CH3/-SH = 6:1 mixed endgroups) surface and 81% for the uncharged-polar SAM (-OH/-SH = 6:1mixed endgroups) surface were collected on the NG1 reflectometer at NIST. These data were analyzed using a new interferometric phasing method employing the neutron scattering contrast between the Si and Fe layers in a single reference multilayer structure and a constrained refinement approach utilizing the finite extent of the gradient of the profile structures for the systems. This provided the water distribution profiles for the two tethered protein monolayers consistent with their electron density profile determined previously via x-ray interferometry (Chupa et al., 1994).

Vectorially-oriented monolayers of detergent-solubilized bovine heart cytochrome c oxidase have been formed by self-assembly from solution and Langmuir-Blodgett (LB) deposition. Both quartz and Ge/Si multilayer substrates, the latter fabricated by molecular beam epitaxy, were alkylated with an amineterminated alkylsiloxane monolayer prior to introduction to the protein. For the self-assembled protein monolayers, the amine end group surface provided for primarily electrostatic interactions with the protein, thereby encouraging a nearly unidirectional vectorial orientation of the so-adsorbed integral membrane protein. This was demonstrated by the analysis of meridional X-ray diffraction data from the monolayers so-adsorbed onto the Ge/Si multilayer substrates, which directly provided electron density profiles of the protein along the axis normal to the substrate plane to a spatial resolution of 10 Å. These profiles are consistent with the three-dimensional structure of the protein, obtained from electron microscopy. Patterson function analysis of meridional X-ray diffraction from the LB-deposited monolayers has shown the profile structure of the so-deposited protein monolayers to be qualitatively similar to that obtained via selfassembly from solution, thereby suggesting that the LB-deposited monolayers are similarly vectoriallyoriented. Optical spectroscopy using quartz substrates has also indicated that the LB monolayers tend to be more densely packed than their self-assembled counterparts. Optical linear dichroism has confirmed that the planes of the oxidase's two heme groups and, hence, the molecule's long axis are more perpendicular to the monolayer plane in the LB case than for the self-assembled monolayers, consistent with the profile length of the molecule along the axis normal to the monolayer plane. Such densely packed, vectoriallyoriented monolayers in a fully hydrated state now provide a unique opportunity to perform directly correlated structural-functional studies on this membrane protein.

Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution. Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cysteine 102 of the yeast cytochrome c. The bimolecular complex was formed by further incubating these cytochrome c monolayers in detergent-solubilized cytochrome oxidase. The sequential formation of such monolayers and the vectorially oriented nature of the cytochrome oxidase was studied via meridional x-ray diffraction, which directly provided electron density profiles of the protein(s) along the axis normal to the substrate plane. The nature of these profiles is consistent with previous work performed on vectorially oriented monolayers of either cytochrome c or cytochrome oxidase alone. Furthermore, optical spectroscopy has indicated that the rate of binding of cytochrome oxidase to the cytochrome c monolayer is an order of magnitude faster than the binding of cytochrome oxidase to an amine-terminated surface that was meant to mimic the ring of lysine residues around the heme edge of cytochrome c, which are known to be involved in the binding of this protein to cytochrome oxidase.

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The version in the Kent Academic Repository may differ from the final published version. Users are advised to check http://kar.kent.ac.uk for the status of the paper. Users should always cite the published version of record.

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