Spectroscopic Investigation of Novel Optogenetic Tools

Optogenetics is a fairly young scientific field that uses the expression of light-sensitive proteins in particular cell types, referred to as optogenetic tools, to modulate their response by light. In this work two different optogenetic tools were investigated. The transmembrane protein KcsA was converted to an artificial light-gated ion channel by an azobenzene photoswitch that carried a tetraethyl ammonium group blocking the ion flow. Interactions of KcsA with different types of photoswitches were investigated by thermophoresis, surfaceenhanced infrared absorption spectroscopy (SEIRAS), and impedance spectroscopy. Microscale thermophoresis (MST) indicated low affinity of KcsA to the majority of probed photoswitches. Thus, a maleimide-labeled azobenzene (MAQ) was bound covalently to the P55C variant of KcsA. Surface-enhanced infrared spectroscopy indicated the successful establishment of a tethered KcsA P55C monolayer reconstituted into lipids. However, impedance spectroscopy did not confirmed the closing and opening of the KcsA channel mediated by the MAQ, although vibrational changes where detected in the SEIRA spectrum. Thus, fast degeneration of the maleimide group probably prevented the reaction with the cysteine of the KcsA and did not lead to a functional artificial system. In the second part of this work, the mechanisms of signal transduction of blue-light photoreceptors containing LOV domains were investigated. Aureochrome 1 from Vaucheria frigida acts as a blue-light activated transcription factor, while mPAC from Microcoleus chthonoplastes is a light activated adenylyl cyclase (AC). The kinetics of the photocycle intermediates LOV715, which is a red-shifted triplet state, and the blue-shifted signaling state LOV390 were determined by time-resolved UV/Vis spectroscopy. In case of aureochrome 1 the presence of the bZIP domain was found to prolong the lifetime of the LOV390 signaling state as compared to the isolated LOV domain, while the binding of DNA did not influence the photocycle kinetics. For mPAC, lifetime of the triplet state was significantly shorter than all other reported LOV domains, while the decay of the adduct state was in the normal range. The light-dark FTIR difference spectra indicated characteristic features of the flavin mononucleotide (FMN) chromophore. Furthermore, the S-H stretching vibration of the reactive cysteines involved in formation of the thio-adduct state have been detected. For aureochrome 1 the frequency was shifted to lower wavenumbers, while in mPAC this band was asymmetric, suggesting the presence of rotational isomers. Furthermore, mPAC showed a negative band that was attributed to an O-H stretch vibration of an amino acid side chain of the adenylyl cyclase domain, which is probably involved in dimerization of this domain. The light-induced FTIR difference spectrum of aureochrome 1 in presence of its target DNA contained vibrations that were assigned to lysine, arginine and phosphate-backbone groups. Thus, a model of the binding complex was determined that shows the critical interactions between the transcription factor and the DNA molecule.