Time-Resolved Fourier Transform Infrared Spectroscopy on Plant Cryptochrome

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Cryptochromes are sensory proteins that act as key players in the regulation of organisms in response to blue light. They set the biological clock in insects and plants and initiate plant development. Furthermore, they act as sensors for the Earth’s magnetic field in insects and putatively in migratory birds. They incorporate oxidized flavin adenine dinucleotide as a chromophore, a derivative of vitamin B2. Absorption of blue light initiates a cascade of events within the protein, most of which have not been resolved yet. As a model system for this project, the photosensitive domain of the plant cryptochrome from the green alga Chlamydomonas is selected. The signalling state of plant cryptochromes is formed by separate transfer of an electron and a proton from the apoprotein to the flavin. We aim at investigating the response of the protein in a time-resolved manner by applying Fourier transform infrared spectroscopy after nanosecond laser excitation. For this purpose, the stepscan methodology needs to be adapted to the specific demands of the cryptochrome system. The response of the apoprotein to the microsecond proton transfer will be monitored and analyzed. Crucial intermediate steps in the photoreaction will be resolved on microsecond and millisecond time scales. The analysis will reveal to which extent proton transfer, secondary structural changes, and redox coupling play a key role in transmission of the signal from the chromophore all the way to the interaction partner.