Stability of purple membranes from Halobacterium salinarum toward surfactants: inkjet printing of a retinal protein.

Inkjet printing is a versatile technique widely applied in biological microarray technology. Because of its photochemical and photophysical properties, bacteriorhodopsin (BR) from Halobacterium salinarum holds promise for applications in nanotechnology, and inkjet printing would simplify the transfer of BR to suitable substrates. Surfactants are essential parts of inkjet formulations tuning viscosity, rheology, and spreading behavior of the solution. However, many surfactants destabilize the structure of proteins and often cause denaturation accompanied by a complete loss of function. Inkjet printing of membrane proteins is particularly challenging and special care must be taken in the choice of the surfactant. For BR, the situation is complicated by the fact that the structural integrity of BR depends on its native membrane environment, the so-called purple membrane (PM). PM contains 10 lipid molecules per BR monomer and is very sensitive toward surfactants. In this work, we identified surfactants suitable for inkjet formulations containing PM. Initially, we screened a variety of technically relevant surfactants for compatibility with PM using the UV-vis absorption of the retinal chromophore as a natural probe. Promising candidates were selected, and their impact on the structure of PM and BR was analyzed using UV-vis spectroscopy, CD spectroscopy, and small-angle X-ray scattering (SAXS). We identified two surfactants compatible with PM and suitable for inkjet formulations. An inkjet formulation containing PM as dye component was developed. We demonstrate that the photochromic properties of BR are maintained upon inkjet printing.

Structural changes in bacteriorhodopsin caused by two-photon-induced photobleaching.

Bacteriorhodopsin (BR) is the key protein of the halobacterial photosynthetic system. BR assembles into two-dimensional crystalline patches, the so-called purple membranes (PM), and acts as a light-driven proton pump converting light energy into the chemical energy of a proton gradient over the cell membrane. The two-photon absorption (TPA) of BR is so far not fully understood. Astonishingly high TPA cross sections have been reported, but the molecular mechanisms have not been elucidated. In this work, we address structural changes in BR and PM upon TPA, investigating its TPA photochemistry by spectroscopy, small-angle X-ray scattering, as well as electron and atomic force microscopy. We observe that TPA of BR leads to formation of an UV-absorbing N-retinyl-bacterioopsin state, which is accompanied by the loss of crystalline order in PM. FTIR and CD spectroscopy confirm that BR trimers as well as the secondary structure of the BR molecules are preserved. We demonstrate that excitation by TPA results in the photochemical reduction of the retinal Schiff base, which in turn causes a permanent asymmetric shape change of BR, similar to the one transiently observed during the photocycle-related opening and closing of the cytoplasmic proton half channel. This shape change causes PM sheets to merely roll up toward the extracellular side and causes the loss of crystallinity of PM. We present a model for the TPA photoresponse of BR, which also explains the irreversibility of the process in terms of a photochemical reduction of the Schiff base.

Crystallinity of purple membranes comprising the chloride-pumping bacteriorhodopsin variant D85T and its modulation by pH and salinity.

Self-assembly of membrane proteins inside the cell membrane critically depends on specific protein-protein and protein-lipid interactions. Purple membranes (PMs) from Halobacterium salinarum comprise wild-type bacteriorhodopsin (BR) and lipids only and form a 2-D crystalline lattice of P3 symmetry in the cell membrane. It is known that removal of the retinylidene residue as well as the exchange of selected amino acids lead to a loss of crystallinity. In PMs comprising the BR variant D85T, we have observed a tunable tendency to form crystalline domains, which depends on pH-value and chloride ion concentration. BR-D85T resembles the function of the chloride pump halorhodopsin. The protonation state of amino acid residues within the binding pocket and chloride binding in the vicinity of the protonated retinal Schiff base affect the overall shape of BR-D85T molecules in the membrane, thereby changing their interactions and subsequently their tendency to form crystalline areas. The combination of small-angle X-ray scattering, atomic force microscopy, and freeze-fracture electron microscopy enables us to analyze the transitions statistically as well as on the single membrane level. PM-D85T is a model system to study membrane protein association upon substrate binding in a native environment. Furthermore, the ability to reversibly modulate the crystallinity of PMs probably will be useful for the preparation of larger artificial crystalline arrays of BR and its variants.