Gesteuerte Assemblierung funktionaler makromolekularer Bausteine: Carbon-NanoMembranen (CNM) und Purpur-Membranen (PM)
Biomaterialien
Zusammenfassung der Projektergebnisse
Purple Membrane (PM) is a semi-crystalline lipid double-layer membrane with a thickness of about 5.2 nm. Due to their high degree of crystallinity, they are highly resistant to chemical and physical influences. PM can survive in highly concentrated salt solutions and with very few water only. Unlike other lipid bilayer membranes PM is rather rigid and not able to fusion. This is due to its high protein content. It consists of single sheets with a size of around 1 µm. Their crystallinity arises from a well-ordered protein lattice solely made of bacteriorhodopsin (BR). BR consists of seven transmembrane α-Helices forming a pore. In this pore, a retinal molecule is bond to the protein. Induced by the light-driven isomerisation of retinal, protons are pumped from the cytoplasmic side to the extracellular side in a multi-step process. BR is arranged as trimers in a hexagonal lattice. As PM consists of BR and lipids only, it has the highest crystalline percentage known to date. Due to its interesting properties a variety of technical applications were proposed. The underlying idea is to take advantage of the proton pump property e.g. for energy conversion or water desalination. In this project, it was tried with various methods to create a PM monolayer onto an ultrathin substrate that is permeable to protons in order to use the so-obtained nanofoil as light-driven proton pump. To choose the most appropriate CNM substrate, the wild type of PM (PM-WT) was sedimented onto the substrate and then washed with distilled water. After drying, the coverage rate was determined. This study was carried out with different PM-WT concentrations and dilution buffers as well as with different incubation times (up to 24 hours). The results manifest 4’-Nitro-1,1’-biphenyl-4-thiol (NBPT) as the best CNM substrate for this purpose. Nevertheless, a largely covering PM-WT monolayer could not be observed. Even though a coverage rate of more than 99 % seems like a good result, the PM rather formed clusters than monolayers. In order to improve the coverage and gain well oriented PM-WT, an electrical field was applied to the drop where the PM was dispersed in. The substrate itself served as one capacitor plate whereas the other plate consisted of indium tin oxide (ITO). The idea was to move PM-WT to areas on the substrate that are not fully covered by PM-WT yet since the electric field is stronger in these unshielded areas. In addition, the PM gets well oriented in an electric field according to its surface charge. For the first attempts, the PM-WT solution drop was attached to the substrate only and was not in contact with the opposite electrode, aiming on field-induced orientation of the PM sheets. The drop was dried on the substrate. For small drops (5 µl) and small PM-WT concentrations good results could be yielded. Higher concentrations and drop sizes lead to cluster building. However, the coverage rate was far away from full coverage. In a further developed process, the PM-WT solution drop was in contact with both electrodes so that a current could flow. The idea behind that is to move the PM-WT sheets not by water evaporation in an external electric field, but rather electrophoretically. Under the right conditions a good quasi-monolayer of PM-WT with very few gaps only could be obtained over comparably huge areas. The experiments were repeated with a genetically modified species of PM (c-His PM) which had histidine tagged to every BR protein and hence was strongly positively loaded on the extracellular side. The histidine tag should bind to the substrate via Ni-NTA linkage. However, instead of single c-His PM sheets, large substrate areas covered with c-His PM were observed. The single sheets merged and fused into one big PM sheet. The detailed mechanism of this phenomenon was investigated by careful variation of the experimental conditions (e.g. voltage, concentration, etc.).
