X-ray interferometry/holography was applied to meridional x-ray diffraction data to determine uniquely the profile structures of a single monolayer of an integral membrane protein and a peripheral membrane protein, each tethered to the surface of a solid inorganic substrate. Bifunctional, organic self-assembled monolayers (SAMs) were utilized to tether the proteins to the surface of Ge/Si multilayer substrates, fabricated by molecular beam epitaxy, to facilitate the interferometric/holographic x-ray structure determination. The peripheral membrane protein yeast cytochrome c was covalently tethered to the surface of a sulfhydryl-terminated 11-siloxyundecanethiol SAM via a disulfide linkage with residue 102. The detergent-solubilized, photosynthetic reaction center integral membrane protein was electrostatically tethered to the surface of an analogous amine-terminated SAM. Optical absorption measurements performed on these two tethered protein monolayer systems were consistent with the x-ray diffraction results indicating the reversible formation of densely packed single monolayers of each fully functional membrane protein on the surface of the respective SAM. The importance of utilizing the organic self-assembled monolayers (as opposed to Langmuir-Blodgett) lies in their ability to tether specifically both soluble peripheral membrane proteins and detergent-solubilized integral membrane proteins. The vectorial orientations of the cytochrome c and the reaction center molecules were readily distinguishable in the profile structure of each monolayer at a spatial resolution of 7 A.
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.
Previous work has recently employed x-ray interferometry for the unique determination of the profile structures of ultrathin Langmuir-Blodgett multilayer films of Cd-arachidate and of tethered protein monolayers on the surface of Ge/Si multilayer substrates [1, 2]. These studies utilized the inorganic substrate as the reference structure for the interferometrie phasing of the meridional x-ray diffraction I (Qxy=0A-1, QZ) from the inorganic-organic composite structure. The substrates, fabricated by magnetron sputtering, contained only broad profile features (≥20 A), thereby limiting the spatial resolution of the organic profile structures so-determined. Molecular beam epitaxy (MBE) permits the fabrication of the multilayer reference structure with profile features as narrow as a single atomic monolayer, thereby providing delta-function-like features in the reference structure. The reference structure can then be tailored such that the autocorrelation function of the inorganic-organic composite profile structure [obtained by a Fourier transform of its meridional diffraction I (Qxy=0A-1, Qz) data without phase information] contains only the organic profile structure itself over a particular range of the profile coordinate z. This approach for uniquely determining the unknown profile structure of the organic overlayer is x-ray holography by analogy to simple off-axis holography with much longer wavelength radiation. We have initially utilized MBE fabricated Ge/Si multilayer substrates of the type N(Ge2Si30), e.g., for N=2 or 3 superlattice unit cells, each containing two Ge monolayers and thirty Si monolayers, to thereby determine the profile structures of four different organic overlayers, namely a) self-assembled alkylsiloxane monolayers, b) Langmuir-Blodgett cadmium alkylcarboxylate monolayers, c) a Langmuir-Blodgett Cd-arachidate head-to-head bilayer deposited on a)-above and, d) a covalently tethered protein monolayer.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.