C4. Signal Transduction by Blue Light Photoreceptors Studied by Time- Resolved Crystallography Keith Moffat, Spencer Anderson, Hyotcherl Ihee, Sudarshan Rajagopal, Vukica Srajer and Reinhard Pahl Department of Biochemistry & Molecular Biology, and BioCARS, The University of Chicago, Chicago, IL 60637, USA Photoreceptors involved in signaling have to harness the energy derived from absorbing a photon and direct it into the formation of a structural signal, manifested either as atomic motion or as altered atomic mobility or both. Further, they have to achieve this with high quantum efficiency, which means that the quantum yields for competing processes such as fluorescence or thermal de-excitation have to be rapidly suppressed. How is this achieved, at the structural level? We address this question by time-resolved crystallographic studies  of the “simple” bacterial blue light photoreceptor, photoactive yellow protein (PYP), over the ns to s time range during which PYP executes a fully-reversible photocycle. PYP is the structural paradigm for the PAS domain family of proteins, which also includes the LOV domain family [2,3]. We study both wild type PYP and its E46Q mutant, in which a key hydrogen bond between the 4-hydroxycinnamic acid chromophore and residue 46 in the surrounding protein is modified; and apply singular value decomposition [4-6] to analyze our time-resolved results. We combine these with static, very high resolution (~1A)  and cryotrapping experiments  to identify the chemical kinetic mechanism and time course of the structural changes during the photocycles [9,10]. These turn out not to be “simple”; rather, they involve the generation of a structural signal in an initially highly-strained chromophore, which progressively relaxes as tertiary structural changes propagate through the protein and ultimately destabilize the N-terminus, ~25A distant from the chromophore. 1. Moffat, K. Chem. Revs. 101, 1569-1581 (2001). 2. Hellingwerf, K., Hendriks, J. & Gensch, T. J. Phys. Chem. A107, 1082-1094 (2003). 3. Crosson, S., Rajagopal, S. & Moffat, K. Biochemistry 42, 2-10 (2003). 4. Schmidt, M. et al., Biophys. J. 84, 2112-2129 (2003). 5. Schmidt., M. et al., PNAS 101, 4799-4804 (2004). 6. Rajagopal, S. et al., Acta Cryst. D60, 860-871 (2004). 7. Anderson, S., Crosson, S. & Moffat, K. Acta Cryst. D60, 1008-1016 (2004). 8. Anderson, S., Srajer, V. & Moffat, K. Photochem. Photobiol., in press (2004). 9. Anderson, S. et al., Structure 12, 1039-1045 (2004). 10. Rajagopal, S. et al., mss. submitted (2004). C-4
C5. Real Time Phase Enhanced Radiography Wah-Keat Lee Advanced Photon Source Argonne National Laboratory 9700 S. Cass Avenue Argonne, IL 60439, USA. Conventional x-ray imaging relies on the differences in the absorption of the sample to provide image contrast. However, with the advent of x-ray sources with small source sizes, such as micro-focus x-ray tubes and synchrotrons, an additional contrast mechanism can come into play, namely, phase contrast. Phase contrast, which includes refraction and diffraction effects, can greatly enhance the image quality. Phase contrast is particularly useful in cases where the absorption contrast is weak. We present comparisons of conventional absorption based images with phase-enhanced images in a variety of samples at different x-ray energies.
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- Spring '19
- X-ray crystallography, Neutron diffraction