A Light-Responsive Self-Assembly Formed by a Cationic Azobenzene Derivative and SDS as a Drug Delivery System.

The structure of a self-assembly formed from a cationic azobenzene derivative, 4-cholesterocarbonyl-4'-(N,N,N-triethylamine butyloxyl bromide) azobenzene (CAB) and surfactant sodium dodecyl sulfate (SDS) in aqueous solution was studied by cryo-TEM and synchrotron radiation small-angle X-ray scattering (SAXS). Both unilamellar and multilamellar vesicles could be observed. CAB in vesicles were capable to undergo reversible trans-to-cis isomerization upon UV or visible light irradiation. The structural change upon UV light irradiation could be catched by SAXS, which demonstrated that the interlamellar spacing of the cis-multilamellar vesicles increased by 0.2-0.3 nm. Based on this microstructural change, the release of rhodamine B (RhB) and doxorubicin (DOX) could be triggered by UV irradiation. When incubated NIH 3T3 cells and Bel 7402 cells with DOX-loaded CAB/SDS vesicles, UV irradiation induced DOX release decreased the viability of both cell lines significantly compared with the non-irradiated cells. The in vitro experiment indicated that CAB/SDS vesicles had high efficiency to deliver loaded molecules into cells. The in vivo experiment showed that CAB/SDS vesicles not only have high drug delivery efficiency into rat retinas, but also could maintain high drug concentration for a longer time. CAB/SDS catanionic vesicles may find potential applications as a smart drug delivery system for controlled release by light.

Crystal structure of Cruxrhodopsin-3 from Haloarcula vallismortis.

Cruxrhodopsin-3 (cR3), a retinylidene protein found in the claret membrane of Haloarcula vallismortis, functions as a light-driven proton pump. In this study, the membrane fusion method was applied to crystallize cR3 into a crystal belonging to space group P321. Diffraction data at 2.1 Å resolution show that cR3 forms a trimeric assembly with bacterioruberin bound to the crevice between neighboring subunits. Although the structure of the proton-release pathway is conserved among proton-pumping archaeal rhodopsins, cR3 possesses the following peculiar structural features: 1) The DE loop is long enough to interact with a neighboring subunit, strengthening the trimeric assembly; 2) Three positive charges are distributed at the cytoplasmic end of helix F, affecting the higher order structure of cR3; 3) The cytoplasmic vicinity of retinal is more rigid in cR3 than in bacteriorhodopsin, affecting the early reaction step in the proton-pumping cycle; 4) the cytoplasmic part of helix E is greatly bent, influencing the proton uptake process. Meanwhile, it was observed that the photobleaching of retinal, which scarcely occurred in the membrane state, became significant when the trimeric assembly of cR3 was dissociated into monomers in the presence of an excess amount of detergent. On the basis of these observations, we discuss structural factors affecting the photostabilities of ion-pumping rhodopsins.

Crystallization and crystal properties of squid rhodopsin.

Rhodopsin, a photoreceptor membrane protein in the retina, is a prototypical member of the G-protein-coupled receptor family. In this study, rhodopsin from the retina of the squid Todarodes pacificus was treated with V8 protease to remove the C-terminal extension. Truncated rhodopsin was selectively extracted from the microvillar membranes using alkyl glucoside in the presence of zinc ions and was then crystallized by the sitting-drop vapour-diffusion method. Of the various crystals obtained, hexagonal crystals grown in the presence of octylglucoside and ammonium sulfate diffracted to 2.8 A resolution. The diffraction data suggested that the crystal belongs to space group P6(2), with unit-cell parameters a = b = 122.1, c = 158.6 A. Preliminary crystallographic analysis, together with linear dichroism results, suggested that the rhodopsin dimers are packed in such a manner that their transmembrane helices are aligned nearly parallel to the c axis.

Direct observations of freeze-etching processes of ice-embedded biomembranes by atomic force microscopy.

We have fabricated a cryogenic atomic force microscope that is designed for structural investigation of freeze-fractured biological specimens. The apparatus is operated in liquid nitrogen gas at atmospheric pressure. Freeze-fracturing, freeze-etching and subsequent imaging are carried out in the same chamber, so that the surface topography of a fractured plane is easily visualized without ice contamination. A controlled superficial sublimation of volatile molecules allows us to obtain three-dimensional views of ultrastructures of biological membranes.