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Abstract
Photoactive yellow protein (PYP) is a bacterial blue-light receptor containing a p-coumaric acid (pCA) chromophore and belongs to the PAS domain superfamily. PYP is a model system to study protein biophysics, particularly protein dynamics, proton transfer, and active site functional tuning, and microbiological processes in the photobiology of bacteria. Here we explore novel PYPs to understand their biophysical and biochemical properties, and their biological functions. With recent advances in DNA sequencing technology, large data sets are available from bacterial genome projects. We identified homologous of the PYP from H. halophila in five bacteria. We cloned and heterologously overproduced these apoproteins in Escherichia coli and reconstituted them to functional PYPs. The results diversify the taxonomic distribution, physiology of bacteria containing PYPs, variation in biophysical functional properties, and amino acid sequences of members of the PYP family. Notable findings on the functional properties of PYPs are that Sr PYP has the most blue-shifted absorption maximum, a very low pCA p Ka, and the slowest known pB decay, and has a 32 amino acid N-terminal extension compared to Hh PYP. Using FTIR spectroscopy, we assigned the Glu46 C=O stretching mode using E46Q Sr PYP. The FTIR spectra confirmed an unusually strong hydrogen bonding interaction between Glu46 and the pCA at the active site of Sr PYP. This hydrogen bond also causes a novel phenomenon, a spectral isotopic effect (SIE) in PYP, in which the absorbance spectrum redshifts in D2O. This SIE was assigned the Glu46-pCA hydrogen bond, and was explained based on the ionic nature of this hydrogen bond. We observed unexpected photostationary partial photobleaching of Sr PYP, and probed the pH and temperature dependence of this phenomenon by UV/Visible absorbance and fluorescence spectroscopy. Our results revealed strong spectral inhomogeneity and complexity in the Sr PYP photocycle. We investigated the biological function of Il PYP and Sr PYP. A novel pharmacological approached revealed that Il PYP is the photoreceptor for light suppression of biofilm formation. Unexpected DNA binding was discovered for Sr PYP, a novel property for a PAS domain protein.
