In-Cell Infrared Difference Spectroscopy on Cryptochromes with Millisecond Time Resolution and in Human Cell Lines

• Applicant: Professor Dr. Tilman Kottke
• Subject Area: Biophysics
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 397169013

Infrared difference spectroscopy on living cells opens up new opportunities to study the mechanisms of receptors in a near-native environment. In the previous funding period we developed the approach for in-cell spectroscopy in transmission and attenuated total reflection configuration on soluble photoreceptors in bacterial cells. Here, we will develop the method further and focus with in-cell spectroscopic methods on cryptochromes, which act as central blue light receptors and key elements of the circadian clock in animals and plants. We will scrutinize in vitro and in cells whether a conserved salt bridge close to the flavin cofactor relays the signal from the photochemical reaction to the response of the protein moiety. The light-induced breaking of the salt bridge might represent a unifying mechanism for cryptochromes. Moreover, we will resolve with in-cell spectroscopy the kinetics of dissociation of the C-terminal extension as a key step in signaling. To this end we will establish time-resolved in-cell infrared spectroscopy with millisecond time resolution for irreversible systems. Finally, we will extend application of infrared difference spectroscopy to human cell lines. Cryptochromes drive liquid-liquid phase separation upon illumination in optogenetic tools and presumably also in nature. We will study these processes in human cell lines in the infrared spectral region under near-native conditions to better understand the conditions in droplets and the photobody formation by cryptochromes. In summary, in-cell infrared difference spectroscopy is a modern tool to explore native receptor mechanisms in living cells. We will extend application of this technique to human cell lines as an expression host with medical relevance and aim to reveal key steps in mechanism and kinetics of signaling pathways within cryptochromes in cells.

• DFG Programme: Research Grants