Chupa J A, McCauley J P, Strongin R M, Smith A B, Blasie J K, Peticolas L J, Bean J C
Department of Chemistry, University of Pennsylvania, Philadelphia 19104.
Biophys J. 1994 Jul;67(1):336-48. doi: 10.1016/S0006-3495(94)80486-2.
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.
X射线干涉测量法/全息术被应用于子午X射线衍射数据，以唯一地确定单个整合膜蛋白和外周膜蛋白单层的轮廓结构，这两种蛋白均连接在固体无机底物表面。利用双功能有机自组装单层膜（SAMs）将蛋白质连接到通过分子束外延制造的Ge/Si多层底物表面，以促进干涉测量/全息X射线结构测定。外周膜蛋白酵母细胞色素c通过与102位残基的二硫键共价连接到巯基封端的11-硅氧基十一烷硫醇SAM表面。去污剂溶解的光合反应中心整合膜蛋白通过静电作用连接到类似的胺封端的SAM表面。对这两个连接的蛋白质单层系统进行的光吸收测量结果与X射线衍射结果一致，表明在各自SAM表面上每个功能完整的膜蛋白形成了紧密堆积的单分子层，且这种形成是可逆的。利用有机自组装单层膜（与朗缪尔-布洛杰特膜相反）的重要性在于其能够特异性地连接可溶性外周膜蛋白和去污剂溶解的整合膜蛋白。在7埃的空间分辨率下，细胞色素c和反应中心分子的矢量方向在每个单分子层的轮廓结构中很容易区分。
