Improvisations in phototrophy. Protein engineering and functional investigation of rhodopsin proton-pumps

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Improvisations in phototrophy. Protein engineering and functional investigation of rhodopsin proton-pumps

Type: Doctoral Thesis
Title: Improvisations in phototrophy. Protein engineering and functional investigation of rhodopsin proton-pumps
Author: Ganapathy, S.
Issue Date: 2017-12-12
Keywords: Phototrophy
Microbial rhodopsin
Proteorhodopsin
Proton pump
Spectral tuning
Retinal analog
Protein engineering
Mutagenesis
Directed evolution microenvironment
Abstract: Microbial rhodopsins are photosensitive pigments implemented in the growth and adaptation of a large population of microorganisms. These relatively simple, tunable photosystems use a molecule of retinal as a chromophore to facilitate the conversion of sunlight to chemical energy. Retinal-based phototrophy is believed to sustain the phototrophic balance of various biospheres and has several important biotechnological applications. In this thesis, we propose the use of microbial rhodopsins as an alternative photosystem in a complementary approach towards more efficient use of photons in the solar spectrum. Towards this end, we describe the adaptation of two rhodopsin proton-pumps, namely proteorhodopsin and Gloeobacter rhodopsin, to shift their action spectrum into the near-infrared region. Several red-shifted variants of were generated by utilizing a combination of retinal analogs with specific opsin mutations. We also constructed a novel directed-evolution set-up, which allows us to generate a library of red-shifted mutants with simultaneous screening for spectral shifts and proton-pumping ability. Finally, the impact of a detergent or lipid microenvironment was tested on the various pigments generated in this study. Our results have important prospects in a number of biotechnological fields such as optogenetics, membrane-sensor technology and as a complementary photosystem for oxygenic photosynthesis.
Promotor: Supervisor: Grip W.J. de, Groot H.J.M. de
Faculty: Science
University: Leiden
Uri: urn:isbn:9789462338265
Handle: http://hdl.handle.net/1887/57985
 

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