The current project is centered on two hypotheses (i) that changes in light emission from the fluorescent proteins that occur under constant illumination result from light-induced changes in the chromophore structure that are controlled by the protein environment and (ii) that the folded fluorescent protein directs and controls the formation of the chromophore. Based on these hypotheses we are (i) determining the changes in chromophore structure following light absorption and (ii) determining the mechanism of chromophore formation. Experiments involve the use of Raman and infrared spectroscopy coupled with other biophysical techniques, such as absorption and fluorescence spectroscopy, to elucidate the changes in chromophore structure that accompany light absorption. In addition, native chemical ligation is being used to insert unnatural amino acids into the chromophore in order to directly probe the mechanism of chromophore formation. A recent highlight of this work is the use of ultrafast vibrational spectroscopy to directly observe proton transfer in the excited state of the protein.

We have extended our studies of fluorescent proteins to include AppA, a BLUF domain transcriptional anti-repressor from Rhodobacter sphaeroides. AppA contains a flavin chromophore, and we are examining how electronic excitation is coupled to structural changes in the protein.  These experiments utilize ultrafast time-resolved fluorescence and vibrational spectroscopy in collaboration with Dr. Steve Meech at UEA, UK.