Absorption of light by the chromophore in the photoreceptor initiates a sequence of chemical reactions. Time-resolved spectroscopy allows to identify these reactions in the protein and their kinetics. We make use of the fact that light-induced changes in structure of chromophore and protein have a characteristic effect on the absorption of the chromophore in the visible range (its color) as well as on the normal vibrations of chromophore and protein.

UV/Vis Spectroscopy

In time-resolved UV/Vis spectroscopy we record the changes in absorption of white light by the chromophore after excitation with a laser pulse with a duration of a few nanoseconds. The whole visible spectrum of the protein sample is recorded as a difference spectrum at many different points in time from nanoseconds to milliseconds after excitation (for an application see Photochem. Photobiol. 2011).

Vibrational Spectroscopy

Fourier transform infrared spectroscopy allows to investigate changes in structure of chromophore and protein and their dynamics (see Angew. Chem. Int. Ed. 2010). Using light-induced difference spectroscopy between light and dark state, we resolve chemical processes of single amino acids and reactions of the chromophore against the background of thousands of normal vibrations of the protein environment. In difference to UV/Vis spectroscopy, reactions can be monitored that take place distant from the chromophore. Additionally infrared spectroscopy is sensitive to changes in secondary and tertiary structure of the proteins.

We apply time-resolved rapid-scan and step-scan techniques to investigate the mechanism of cyclic processes over a broad time range from microseconds to seconds after excitation with a pulsed laser (see Biophys. J. 2009). We are further developing these methods to enable investigation of systems with long cycling times and irreversible reactions. (see PCCP 2013).

Theory

For interpretation and assignment of the infrared spectra, we also make use of quantum chemical calculations including models for the environment. Even difference spectra can be calculated (see J. Phys. Chem. Lett. 2010, PCCP 2013).

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