Photoactive yellow protein (PYP) is the prototype for a large family of proteins containing a structural motif referred to as the PAS domain. PAS domains function in a variety of signaling and regulatory roles in eucaryotes as well as procaryotes. Photoactive yellow protein (PYP) is a small soluble protein which appears to have a variety of signaling roles in purple bacteria. The goals of the proposed studies are four fold. 1. To elucidate the chemical and structural characteristics of the photocycle intermediates. 2.To understand the role of specific amino acid side chains in the kinetic and spectral properties of the ground state and intermediates. 3. To establish the mechanism(s) by which PYP signals to its reaction partners and utilizes the photocycle to function in vivo. 4. To establish the differences, in both functional and physico-chemical properties, among the three types of PYP identified to date. Structural, thermodynamic, kinetic and optical properties of the wild-type, site-specific mutants and variant chromophores will be studied. A variety of techniques will be utilized, including x-ray crystallography, NMR, UV/VIS spectroscopy, kinetic measurements in time domains ranging from femtoseconds to seconds, picosecond Coherent Anti-Stokes Raman Spectroscopy (CARS), picosecond time resolved CARS, and computer modeling.The significance of the proposed work is three fold. First, PYP will provide key insights which will help elucidate the role of PAS domains in signaling and regulation. This information will contribute directly to a better understanding of all of the members of the PAS domain family. Second, PYP offers the opportunity to understand the key elements in photoactive proteins - the spectral red-shift of the ground state protein and of key intermediates, how photoisomerization can be used to drive function, and the structure of photocycle intermediates. This level of understanding will complement work with membrane-bound photoactive proteins (bacteriorhodopsin, sensory rhodopsin and rhodopsin) and will provide information not available with these systems. Third, it is possible that an understanding of PYP and photoactive proteins in general will contribute to the development of photobioelectronic switching devices which will have applications in the next generation of small, fast, low power electronics
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