|Bielefeld University||Department of Chemistry||Physical and Biophysical Chemistry||deutsch|
Time-Resolved FTIR and UV/Vis Spectroscopy
Absorption of light by a chromophore initiates a sequence of chemical reactions with the environment. Time-resolved spectroscopy allows us to identify these reactions and their kinetics. We make use of the fact that light-induced changes in structure of the chromophore have a characteristic effect on its absorption in the visible range (its color) as well as on its vibrations (its normal modes).
In photosensors, the chromophore reacts with amino acids in the protein environment. These amino acids can be identified by applying spectroscopy. Additionally, vibrational spectroscopy allows us to elucidate changes in secondary and tertiary structure of proteins induced by the photoreaction.
Time-Resolved UV/Vis Spectroscopy
In time-resolved UV/Vis spectroscopy we record 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 60 nanoseconds to milliseconds after excitation (see Photochem. Photobiol. 2011). Data are analyzed by singular value decomposition and global fit, which yields a kinetic model of the reaction.
Fourier transform infrared spectroscopy (FTIR) allows us to investigate changes in structure of chromophores and proteins and their dynamics (see Angew. Chem. Int. Ed. 2010). Using light-induced difference spectroscopy between light and dark state, we resolve reactions of chromophores and chemical processes of single amino acids against the background of thousands of normal vibrations of the (protein) environment.
FTIR spectroscopy has several advantages compared to UV/Vis spectroscopy.
The smaller line width and the larger number of transitions results in a much higher specificity and thereby a clear identification.
Several additional amino acids can be detected that do not absorb in the UV/Vis region.
Furthermore, only infrared spectroscopy is sensitive to changes in secondary and tertiary structure of proteins.
We achieve an assignment of signals to specific secondary structural elements by
performing segment-resolved double difference spectroscopy (see Biochemistry 2010).
Time-Resolved Vibrational Spectroscopy
We apply and develop time-resolved rapid-scan and step-scan techniques to investigate the mechanism of cyclic processes over a broad time range from 2 microseconds to seconds after excitation with a pulsed laser (see Biophys. J. 2009). The goal of method development is to enable investigation of systems with long cycling times and irreversible reactions. Among other approaches, this goal is achieved by integrating novel flow cells (see PCCP 2013).
The stepscan method covers an important time range, which is not at all or only in principal accessible by other IR techniques auch as ultrafast pump-probe spectroscopy. The recording in stepscan is performed continuously with respect to both frequency and time and thereby leads to a real 3D information.
For interpretation and assignment of the infrared spectra, we also perform quantum chemical calculations including models for the environment. Even difference spectra can be calculated (see J. Phys. Chem. Lett. 2010, PCCP 2013).