Proteo-Lipobeads : a novel platform to investigate strictly oriented membrane proteins in their functionally active form. Bio-UV-SPR: exploring the ultraviolet spectral range for water-bound analytes in surface plasmon resonance spectroscopy
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Abstract
The present thesis comprises two independent parts, the first one (Proteo-Lipobeads) representing approximately 80 % of the work, whereas the remaining 20 % are covered by the second topic (Bio-UV-SPR).
Membrane proteins, which need to be embedded in cell membranes or biomimetic membrane systems to provide function, are the target of many diseases. In the present work, proteo-lipobeads, which overcome the disadvantages of cells, liposomes, and solid-supported bilayer lipid membranes are introduced as a complementary system that enlarges the spectrum of possible investigation methods.
Proteo-lipobeads consist of spherical core particles determining their final size, the proteins of interest, which are oriented by attachment via his-tag technology, and a protein-tethered membrane, which is assembled by detergent removal via dialysis in the presence of the desired lipids. In this work, model proteins of the respiratory chain, cytochrome c oxidase and the cytochrome bc1 complex, are investigated.
The results clearly indicate that the established membrane is dense, the proteins retain their functionality, and that the proteo-lipobead components constitute a modular system. The applicability in high-throughput assays, a certain storage stability, and the possibility to integrate systems that are more complex is evidenced. Besides the micrometer-sized agarose-based proteo-lipobeads, investigated by fluorescence microscopy, a nanometer-sized version is introduced using silica particles to enable UV/Vis spectroscopy. The results are documented by four publications.
In the second part, the commonly used surface plasmon resonance spectroscopy setup was improved to overcome limitations that preclude analyses of water-bound samples at absorption maxima below 350 nm. The answer to this problem is an aluminum grating excited by a white light source as demonstrated on a model protein. The results are documented in a research article.