Table of Contents:
In this disputation techniques were elaborated which allow the structuring of purple membranes on solid substrates. Particularly the morphology of PM on different substrates was investigated and the orientation of the membranes and the fusion of PM to produce extended PM-monolayers were realized.
The bending of the Bacteriorhodopsin mutants D85N and D85T in alkaline solutions were examined by means of atomic force microscopy and single molecule force spectroscopy. The results confirm the model that M-state formation during the photocycle of BR causes an opening of the cytoplasmic half-channel which induces large scale bending of the membranes. The bending direction of the D85X membranes was identified by single molecule force spectroscopy and it was shown that the forces, which cause the bending of the membranes, are stronger than the interactions between PM and surface.
A new method to immobilize PM on surfaces in an oriented way was developed in this work. Genetically modified BR-Q3C which contains a thiole group in the N terminus was bound on ultrasmooth gold substrates. Because the N-terminus of BR-Q3C was accessible only from the extracellular membrane side, the membranes could be bound with their extracellular side covalently to the gold surface. The unspecifically bound oppositely oriented PMs could be removed, so that a highly oriented PM-monolayer was received. The side of the bound PM could be identified by single molecule force spectroscopy. The results concluded a high degree of orientation of the PMs on the gold surface. The force curves taken on BR-Q3C showed an additional Peak which was caused by the gold-Cysteine connection. The unfolding of the Helix A could be analyzed for the first time with force spectroscopy experiments. Another result arose from the analysis of the force curves: The transmembrane α-Helices of some BR-monomers were not unfolded pairwise. These force curves resulted from unfolding the Helices A-C in a simultaneous process.
To fuse purple membranes on surfaces the influence of different substrates on the morphology of the membranes was examined. Surprisingly it was found that PM seems not to be structurally stable in general when adsorbed to a surface. On mica, which is used for most of the AFM imaging experiments with PM, time- and temperature-dependent denaturation was observed. The progression of the denaturation could be observed with molecular resolution.
The denaturation could be reduced by adjusting the interactions between PM and the support. Polyaspartic acid-coated mica and the substrates gold and silicon had a lower denaturating effect. The modified mica as well as gold and silicon were covered only sparsely with PM. The attractive and repulsive forces which take effect on adsorbed PM on surfaces are discussed in chapter 3.3.1. On the one hand these forces can lead to the denaturation of the sample; on the other hand they are necessary to hold the PM on the surface. It became apparent that only on untreated mica surfaces a sufficient high degree of coverage with PM was reached which allows the fusion of the purple membranes to a continuous monolayer.
Bleached purple membranes were adsorbed on mica and were regenerated with retinal. This approach led to a polycrystalline monolayer. 50-100 nm sized crystalline areas were imaged by AFM investigations with molecular resolution. Thereby the crystal structures of the ordered membrane parts were found to be distorted in comparison to each other. Time-dependent analyses showed the mobility of the areas within the membrane.
Improved methods to regenerate PM on mica surfaces were developed, while several factors, which had an effect on the regeneration of the membranes, were examined. It turned out that the ethanol which was used as a solvent of the retinal had a denaturating effect on the membranes. The regeneration without addition of retinal led to crystalline areas with a diameter of several hundred nanometers. With the improved methods approx. 2/3 of the surface occupied with PM could be regenerated.
The results obtained in this work are relevant for many applications of Bacteriorhodopsin in which an orientation of PM or a contact of PM-monolayers with a substrate is necessary. A solution for the problem of the denaturation of PM was found by influencing of the surface interaction. Nevertheless, beside the denaturation the degree of coverage and the desorption of the membranes have to be considered. In general a suitable substrate must be chosen for interfaces between biomolecules and established technology, while all these factors are regarded. Further investigations of the complex interactions on surfaces are of interest to many areas of biotechnology - also proceeding improvement of the fusion of native PM could benefit from the knowledge gathered here.