Receptor activation is at the basis of all biological signal transduction events. Malfunctioning of signaling pathways can cause cancer. We study the mechanism of receptor activation using photoactive yellow protein (PYP), a PAS domain photoreceptor, as a powerful model system. PYP exhibits rhodopsin-like photochemistry based on its p-coumaric acid (pCA) chromophore, and has been investigated by X-ray crystallography. We have recently shown that the presence of a protein crystal lattice greatly reduces the structural changes that occur during the formation of the pB photocycle intermediate, the presumed signaling state of PYP. In addition, our preliminary results strongly indicate that the pB intermediate is partially unfolded. In this proposal we will examine the role of partial protein unfolding upon receptor activation as a novel signal transduction mechanism. (i) We will establish the extent of partial protein unfolding during pB formation by NMR spectroscopy, and by two novel methods: light-induced H/D exchange and light-induced differential scanning calorimetry. In addition, we will examine the hypothesis that the pB intermediate is a molten globule state by studying changes in ANS fluorescence, changes in CD spectrum, and changes in the radius of gyration upon pB formation. (ii) We will examine the effects of the presence of a crystal lattice on the kinetics and thermodynamics of the photocycle using both wt- PYP and the E46Q mutant in both P6 3 and P6 5 crystals by flash photolysis at a range of temperatures. (iii) We will test the hypothesis that the Glu46-pCA couple constitutes the built-in default for the light-triggered protein quake in PYP. These experiments will reveal changes in functional dynamics imposed by a crystal lattice, and will determine the role of transient partial protein unfolding as a novel signal transduction mechanism that is anticipated to play a role not only in PYP but also in other signal transduction systems.
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