Structure-function relationship in photoactive yellow protein (PYP) from Ectothiorhodospira halophila

Abstract

How does a biological light sensor convert the energy of a photon through a sequence of structural changes to generate a biological signal? Photoactive Yellow Protein (PYP) isolated from the phototrophic bacterium Ectothiorhodospira halophila, a small water-soluble protein whose three-dimensional X-ray crystallographic structure has been determined to high resolution, serves as a paradigm for structural studies of the interaction of light and proteins. This blue light photosensor has been implicated in the negative phototactic response of these bacteria. PYP undergoes a cyclic series of absorbance changes upon illumination at its λ(max) of 446 nm. In its ground state, the anionic p-hydroxycinnamoyl chromophore of PYP is covalently bound as a thiol ester to Cys69, buried in a hydrophobic pocket, and hydrogen bonded via its phenolate oxygen to Glu46 and Tyr42. The chromophore becomes protonated in the photobleached state (I₂) after it undergoes trans-cis isomerization, which results in breaking of the H-bond between Glu46 and the chromophore and partial exposure of the phenolic ring to the solvent. To gain an in-depth understanding of these interactions at the molecular level, the active site of the protein and the chromophore structure was modulated via site-directed mutagenesis and incorporation of variant chromophores. The structural, optical, kinetic and thermodynamic properties of several such altered proteins have been investigated and presented in this dissertation. Interestingly, Glu46Asp and Glu46Ala mutations demonstrated dual photoactive species as a result of a pH driven color transition. Met100Ala was the first PYP mutant to exhibit properties of an optical switch. The unique properties of PYP and its mutant forms may eventually permit their use in optical devices for switching, memory, computing and holographic applications. Early stages of the photocycle were characterized using picosecond and femtosecond ultrafast transient absorption spectroscopy. These time-resolved spectroscopic studies have revealed the presence of two new intermediates. The time constants for formation and decay of these intermediates have now been resolved and the structural and mechanistic aspects of these results are discussed. Recently, PYP was proposed as a structural prototype for the PAS domain superfamily. PYP/PAS domains therefore form an important structural motif for biological signaling.