Vectorially Oriented Monolayers of the Cytochrome c/Cytochrome Oxidase Bimolecular Complex

Abstract

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.

Hydration State of Single Cytochrome c Monolayers on Soft Interfaces via Neutron Interferometry

“…6 to be on the order of 20 -25% for the nonpolar and unchargedpolar SAM cases. However, the area per cytochrome c molecule for both SAM surfaces has been consistently found to be ϳ1000 Å 2 /molecule using optical absorption spectroscopy and optical linear dichroism (Edwards et al, 1998). If, instead, we therefore assume this value for the area per molecule obtained independently from the optical absorption spectroscopy, we can then utilize the following expression for the difference neutron scattering density profile for partial hydration with D 2 O versus H 2 O containing the one unknown N water :…”

“…The highly constrained box-refinement approach to interferometric phasing therefore applies an additional constraint on the data to provide a unique profile structure that correctly predicts the experimentally observed reflectivity. Our group has used this method successfully to solve for the electron density profile structures for a variety of fatty acid and protein ultrathin films on solid substrates (Murphy et al, 1993;Chupa et al, 1994;Prokop et al, 1996;Edwards et al, 1998). In this paper we use it to solve for the neutron scattering length density profile of a tethered protein ultrathin film.…”

“…Previous optical spectroscopy studies have shown that yeast cytochrome c (YCC) covalently bound to a soft interface and partially hydrated by a moist helium atmosphere at sufficiently high relative humidity can be fully functional with respect to the oxidation-reduction chemistry of its iron porphyrin prosthetic group and reversibly unbound to large extent without loss of function (Pachence et al, 1990;Pachence and Blasie, 1991;Chupa et al, 1994;Edwards et al, 1998). Water of hydration is important for maintaining the structure of soluble proteins, including the membrane protein YCC, so it would be interesting to know how much water hydrates each YCC molecule in such a monolayer as required to maintain protein function.…”

New Developments for the Site-Specific Attachment of Protein to Surfaces

“…This apparent instability was very likely due to the increased hydrophobic character of the surface. Edwards et al 49 used in this case the naturally occurring thiol group of the Cys102 of yeast cytochrome c for the creation of a vectorially oriented monolayer of the bimolecular complex between yeast cytochrome c and bovine heart cytochrome c oxidase on both quartz and Ge/Si multilayer substrates. In both cases the surfaces were silanized with APS (see Fig.…”

Polarized XAS on vectorially oriented single monolayers of cytochrome c

“…Langmuir-Blodgett) monolayer systems that were not structurally characterized (Song et al, 1993;Cullinson et al, 1994;Owaku et al, 1995;Jiang et al, 1996;Guo et al, 1996), we have focused instead on developing the physical techniques essential to determining the key structural features of the proteins within such vectorially oriented single monolayers. To date, this work has included resonance x-ray diffraction (Pachence et al, 1989), optical linear dichroism (Pachence et al, 1990), and x-ray interferometry/holography (Blasie et al, 1992;Chupa et ai., 1994;Edwards et al, 1997 andEdwards et al, 1998). We have further demonstrated the potential of applying polarized XAFS to such systems in order to obtain information on both the orientation of the protein with respect to the substrate and the detailed structure around the metal site(s) of the protein (Zhang et al, 1997).…”