Part 1: Biochemical and photochemical characterization of the Listeria monocytogenes LOV-STAS protein. Part 2: The possible role of structural water clusters as the proton acceptor in the adduct decay of oat phototropin 1 LOV2 domain

Part 1 of my work began with the genome analysis of the pathogen Listeria monocytogenes. We discovered that there is a blue-light photoreceptor LOV protein coupled to the STAS domain (LM LOV-STAS), which is known to be one of the proteins involved in the bacterial survival system. It is homologous to YtvA, another LOV-STAS blue-light receptor from soil bacteria Bacillus subtilis. Yet, the one from L. monocytogenes has never been studied. A natural question that arised was the function of this protein. Because it is a photoreceptor in a pathogen, can there be correlation between photochemistry and virulence and/or survival in the host, as it was found in Brucella? My thesis is to investigate the photochemistry of the species. I characterized basic biochemical features of the protein, and found that it, like the other photoreceptor LOV domains reported, LM LOV-STAS also contained the chromophore FMN. The protein also undergoes a photocycle upon blue-light illumination, but with a much slower kinetics.;Part 2 is a more detail analysis of an earlier observation in our laboratory and some published findings: dehydration of the LOV domain resulted in a slower adduct decay kinetics. However, it is not known whether it is bulk (free) water or structural (bound) water that makes the adduct decay possible. I used UV-visible and FTIR spectroscopy to investigate the correlation between the effect of dehydration level on the adduct decay kinetics, and the number of hydrogen-bonded water per flavoprotein. My results indicate that an intramolecular water cluster of strongly hydrogen-bonded water near the chromophore my serve as the primary proton acceptor in the proton transfer reaction in the oat phot1 LOV2 adduct decay mechanism.