FTIR Spectroscopy of Red and Far-Red-Light Sensing Phytochromes In vitro

The action of phytochromes is controlled by a tetrapyrrole bilin chromophore that isomerizes upon red or far-red-light illumination. Photoactivation results in a multitude of structural rearrangements of the protein, including secondary structural changes and changes in the interaction patterns of the chromophore and the protein scaffold. The IR signals of phytochromes originate from molecular vibrations of both the chromophore and the protein. Being sensitive to molecular interactions, the vibrational frequencies reveal sub-angstrom-level information about structural changes of a phytochrome protein in solution. Phytochromes are relatively easy to produce and modify, they can be concentrated into high concentrations, and their photocycle is reversible, both thermally and by light. These qualities make phytochromes excellent molecules for FTIR spectroscopic studies. Furthermore, the effect of pH, temperature, or buffer conditions on photoactivation or intramolecular interactions can be studied by means of FTIR spectroscopy.