Vectorially oriented membrane protein monolayers: profile structures via x-ray interferometry/holography.
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.
Chupa
J A
JA
Department of Chemistry, University of Pennsylvania, Philadelphia 19104.
McCauley
J P
JP
Strongin
R M
RM
Smith
A B
AB
Blasie
J K
JK
Peticolas
L J
LJ
Bean
J C
JC
D000588:Amines; D014450:Electron Transport Complex III / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000737:chemistry; D006696:Holography / Q000379:methods; D007368:Interferometry / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry*; D011487:Protein Conformation; D012242:Rhodobacter sphaeroides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D012821:Silanes; D013438:Sulfhydryl Compounds; D014961:X-Ray Diffraction
D000588:Amines; D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D012821:Silanes; D013438:Sulfhydryl Compounds; D003576:Electron Transport Complex IV; D014450:Electron Transport Complex III