Fusion of purple membranes triggered by immobilization on carbon nanomembranes

A freestanding ultrathin hybrid membrane was synthesized comprising two functional layers, that is, first, a carbon nanomembrane (CNM) produced by electron irradiation-induced cross-linking of a self-assembled monolayer (SAM) of 4′-nitro-1,1′-biphenyl-4-thiol (NBPT) and second, purple membrane (PM) containing genetically modified bacteriorhodopsin (BR) carrying a C-terminal His-tag. The NBPT-CNM was further modified to carry nitrilotriacetic acid (NTA) terminal groups for the interaction with the His-tagged PMs forming a quasi-monolayer of His-tagged PM on top of the CNM-NTA. The formation of the Ni-NTA/His-tag complex leads to the unidirectional orientation of PM on the CNM substrate. Electrophoretic sedimentation was employed to optimize the surface coverage and to close gaps between the PM patches. This procedure for the immobilization of oriented dense PM facilitates the spontaneous fusion of individual PM patches, forming larger membrane areas. This is, to our knowledge, the very first procedure described to induce the oriented fusion of PM on a solid support. The resulting hybrid membrane has a potential application as a light-driven two-dimensional proton-pumping membrane, for instance, for light-driven seawater desalination as envisioned soon after the discovery of PM.

Engineering and Production of the Light-Driven Proton Pump Bacteriorhodopsin in 2D Crystals for Basic Research and Applied Technologies

The light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum is a retinal-binding protein, which forms highly ordered and thermally stable 2D crystals in native membranes (termed purple membranes). BR and purple membranes (PMs) have been and are still being intensively studied by numerous researchers from different scientific disciplines. Furthermore, PMs are being successfully used in new, emerging technologies such as bioelectronics and bionanotechnology. Most published studies used the wild-type form of BR, because of the intrinsic difficulty to produce genetically modified versions in purple membranes homologously. However, modification and engineering is crucial for studies in basic research and, in particular, to tailor BR for specific applications in applied sciences. We present an extensive and detailed protocol ranging from the genetic modification and cultivation of H. salinarum to the isolation, and biochemical, biophysical and functional characterization of BR and purple membranes. Pitfalls and problems of the homologous expression of BR versions in H. salinarum are discussed and possible solutions presented. The protocol is intended to facilitate the access to genetically modified BR versions for researchers of different scientific disciplines, thus increasing the application of this versatile biomaterial.

Cells Cultivation System and Preparation of the Purple Membranes for Bionanotechnology

ABSTRACT: The purple membranes (PM) of the Halobacterium salinarum cells and bacteriorhodopsin (major retinal-containing trans-membrane protein of these bacteria are the most promising photosensitive biopolymers in terms of bionanotechnology, developing photosensitive devices, etc. The article is devoted to the improvement of the cultivation system for Halobacterium salinarum cells and the PM production; the study of the properties and functional characterization of bacteriorhodopsin - the biological components of the bionanomaterials. The optimal conditions for the production of the biomass are the following: the volume of incubation media must be 10% of the working volume (the volume of the cuvette); the cell “age” must be close to the end of the exponential phase of growth; the lighting must be maximum (4 light bulbs LD-20); the temperature must be 37°С; the time must be 8 days; the aeration must be 0.45 (L air)/(L of environment)/min. The yield of the product is about 5 g of biomass containing 45 mg of bacteriorhodopsin. The purple membranes and their major protein - bacteriorhodopsin were obtained with the required parameters.