Our understanding of reaction mechanisms in biological systems at the molecular level is presently based largely on knowledge of static, chemically- or physically-trapped structures obtained by high resolution X-ray crystallographic and NMR techniques. However, the dynamic aspects of changes in structure are critical in all chemical and biological processes, for example catalysis, ligand binding and release, and signal transduction. To explore the mechanisms of signal transduction, we have successfully conducted time-resolved crystallographic experiments with ~100 picosecond time resolution and high crystallographic resolution on light-sensitive systems, identified the structures of short-lived intermediates and characterized the overall mechanism of light-dependent signal transduction which these intermediates populate. Recently, we have determined the static crystal structures of several novel, naturally-occurring, signaling photoreceptors which are based on a modular architecture. Those containing so-called LOV or BLUF sensor domains respond to blue light, and bacteriophytochromes containing PAS-GAF-PHY domains to red/far-red light. We will now conduct time-resolved crystallographic experiments on these proteins, and on longer constructs that contain both sensor and effector (output) domains. We address the questions: How is a signal generated and transmitted? How is activity controlled by light? In each, what is the mechanism of light-dependent signal transduction? Despite the high chemical and structural diversity in natural photoreceptors, we believe that general principles of signal transduction exist. Indeed, we have prepared chimeric, artificial photoreceptors in which we have made light-sensitive a normally light-inert biological activity e.g. histidine kinase or DNA binding. We will explore these artificial photoreceptors to test and expand our general principles, afford useful tools and offer new targets for crystallization. Finally, artificial photoreceptors overcome a limitation of time-resolved crystallography: many interesting systems are not light-dependent. We will pursue the question: How general are these approaches?

Public Health Relevance

Many cancers are associated with derangement of signal transduction pathways, driven by ligand binding to chemoreceptors. The pathways may be known but the mechanisms at the molecular level are not. The natural and artificial photoreceptors whose molecular mechanisms we study have parallels to, but also key differences from, chemoreceptors: the thermodynamic and structural principles of signal transduction are likely to be similar in both.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function C Study Section (MSFC)
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Flicker, Paula F
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University of Chicago
Schools of Medicine
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Yang, Xiaojing; Stojkovi?, Emina A; Ozarowski, Wesley B et al. (2015) Light Signaling Mechanism of Two Tandem Bacteriophytochromes. Structure 23:1179-89
Moffat, Keith (2014) Time-resolved crystallography and protein design: signalling photoreceptors and optogenetics. Philos Trans R Soc Lond B Biol Sci 369:20130568
Stojkovi?, Emina A; Toh, K C; Alexandre, Maxime T A et al. (2014) FTIR Spectroscopy Revealing Light-Dependent Refolding of the Conserved Tongue Region of Bacteriophytochrome. J Phys Chem Lett 5:2512-2515
Jung, Yang Ouk; Lee, Jae Hyuk; Kim, Joonghan et al. (2013) Volume-conserving trans-cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography. Nat Chem 5:212-20
Nieder, Jana B; Stojkovi?, Emina A; Moffat, Keith et al. (2013) Pigment-protein interactions in phytochromes probed by fluorescence line narrowing spectroscopy. J Phys Chem B 117:14940-50
Halavaty, Andrei S; Moffat, Keith (2013) Coiled-coil dimerization of the LOV2 domain of the blue-light photoreceptor phototropin 1 from Arabidopsis thaliana. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:1316-21
Sugishima, Masakazu; Moffat, Keith; Noguchi, Masato (2012) Discrimination between CO and O(2) in heme oxygenase: comparison of static structures and dynamic conformation changes following CO photolysis. Biochemistry 51:8554-62
Ohlendorf, Robert; Vidavski, Roee R; Eldar, Avigdor et al. (2012) From dusk till dawn: one-plasmid systems for light-regulated gene expression. J Mol Biol 416:534-42
Mitra, Devrani; Yang, Xiaojing; Moffat, Keith (2012) Crystal structures of Aureochrome1 LOV suggest new design strategies for optogenetics. Structure 20:698-706
Neutze, Richard; Moffat, Keith (2012) Time-resolved structural studies at synchrotrons and X-ray free electron lasers: opportunities and challenges. Curr Opin Struct Biol 22:651-9

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