Titel der Dissertation:
Exploiting Photoisomerization: Spectroscopy on a Carotenoid Sensor and Retinal Proteins
Exploiting Photoisomerization: Spectroscopy on a Carotenoid Sensor and Retinal Proteins
Abstract: Light-based methodologies enjoy popularity due to their non-invasive nature. In particular in the field of optogenetics, where genetic targeting of neurons permits not only simultaneous imaging of a large number of cells but also optical control of neuronal activity. For this, ion channels or pumps are inserted into the membrane which are activated by light. A deep biophysical understanding of the optogenetic systems is key for their successful application.
In a pioneering approach we combined time-resolved absorption spectroscopy with serial femtosecond X-ray crystallography to scrutinize mechanistic details of sodium pumping in Krokinobacter eikastus rhodopsin 2 (KR2). Using an infrared-emitting quantum cascade laser (QCL), we verified that crystalline KR2 exhibits reaction kinetics similar to those observed in its detergent solubilized form. Hereupon, we have identified a previously proposed transient sodium binding site during the O intermediate where the sodium is coordinated by the amino acid side chains of N112 and D251. The findings regarding the ion transport mechanism in KR2 will facilitate the design of protein variants for an optogenetic application.
Bistable G-protein coupled receptors (GPCRs) have two thermally stable conformations and are a promising class of rhodopsins which have the potential to serve as optogenetic switches. I was able to conduct a first biophysical characterization of the invertebrate jumping spider rhodopsin-1 (JSR1). I propose a model of the two-photon reaction based on spectroscopic results. A proposed mediating water molecule as part of the counterion complex in the inactive conformation is identified by Raman spectroscopy and later confirmed by an X-ray crystallographic structure.
In conclusion, these findings provides insights into the mechanistic details of established and upcoming optogenetic tools. This will help to adapt their biophysical properties better suiting the needs of application.
In a pioneering approach we combined time-resolved absorption spectroscopy with serial femtosecond X-ray crystallography to scrutinize mechanistic details of sodium pumping in Krokinobacter eikastus rhodopsin 2 (KR2). Using an infrared-emitting quantum cascade laser (QCL), we verified that crystalline KR2 exhibits reaction kinetics similar to those observed in its detergent solubilized form. Hereupon, we have identified a previously proposed transient sodium binding site during the O intermediate where the sodium is coordinated by the amino acid side chains of N112 and D251. The findings regarding the ion transport mechanism in KR2 will facilitate the design of protein variants for an optogenetic application.
Bistable G-protein coupled receptors (GPCRs) have two thermally stable conformations and are a promising class of rhodopsins which have the potential to serve as optogenetic switches. I was able to conduct a first biophysical characterization of the invertebrate jumping spider rhodopsin-1 (JSR1). I propose a model of the two-photon reaction based on spectroscopic results. A proposed mediating water molecule as part of the counterion complex in the inactive conformation is identified by Raman spectroscopy and later confirmed by an X-ray crystallographic structure.
In conclusion, these findings provides insights into the mechanistic details of established and upcoming optogenetic tools. This will help to adapt their biophysical properties better suiting the needs of application.
Zeit & Ort
10.12.2020 | 15:30