A complete understanding of biological function at the molecular level requires an understanding both of static structure, and of the changes in structure that occur during processes such as enzyme catalysis, ligand binding and release, photocycling, and protein unfolding/refolding reactions. These changes in structure typically occur very rapidly under physiological conditions; the lifetimes of functionally crucial, structural intermediates typically lie in the range from seconds to microseconds. Such short-lived, intermediate structures are completely inaccessible to conventional x-ray crystallographic approaches. With the advent of very intense, polychromatic synchrotron x-ray sources, static x-ray diffraction patterns can be recorded from single crystals of strongly scattering proteins in the one to hundreds of milliseconds range, using a sensitive Kodak storage phosphor area detector. Advances now being made in the x-ray source itself, and in focussing optics, will reduce these exposure times by one to two orders of magnitude into the tens of microseconds to one millisecond range. If the detector is moved in its plane during the exposure, the Laue diffraction spots become streaks; and if the structure of the molecules in the crystal lattice changes during the exposure, then the intensities of the Laue diffraction streaks change, and constitute the raw data of a time-resolved x-ray crystallographic experiment. This experiment has three components, reaction initiation, in which a structural perturbation is induced, reaction monitoring and data acquisition, in which the x-ray intensities are measured as a function of time after reaction initiation, and data analysis, in which the time-dependent changes in molecular structure are identified. We propose to apply time-resolved crystallography to three main systems: the photocycle of photoactive yellow protein that appears to be a simple bacterial photosensor; the photolysis of carboxymyoglobin at low temperature; and the early stages of protein unfolding in the crystal lattice, induced by a temperature jump. The laser techniques necessary for reaction initiation and optical monitoring of the crystals will be further developed. Data analysis strategies aimed at revealing the structures of individual intermediates, rather than just their time-dependent mixture, will be explored.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM036452-05
Application #
3290456
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1990-09-01
Project End
1994-11-30
Budget Start
1990-09-01
Budget End
1990-11-30
Support Year
5
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Chicago
Department
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
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
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
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
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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