XIII European Conference on the Spectroscopy of ... - ecsbm 2009

Palermo, August 28 – September 2, 2009 13 th ECSBM −−−− Book of Abstracts
Protein structural dynamics visualized by time-resolved
X-ray crystallography and liquidography
H. IHEE
Center for Time-Resolved Diffraction, Department of Chemistry, KAIST,
305-701, KOREA hyotcherl.ihee@kaist.ac.kr
The principle, experimental technique, data analysis, and applications of time-resolved X-ray
diffraction and scattering to study spatiotemporal reaction dynamics of proteins in single crystals
and solutions will be presented. X-ray crystallography, the major structural tool to determine 3D
structures of proteins, can be extended to time-resolved X-ray crystallography with a laserexcitation
and X-ray-probe scheme, and all the atomic positions in a protein can be tracked during
their biological function. A study using time-resolved X-ray crystallography will be presented for
the photocycle of the photoactive yellow protein (PYP). Although time-resolved X-ray
crystallography has realized the 3D visualization of protein structural dynamics, its applicability
has been limited to a few model systems with reversible photocycles due to the stringent
prerequisites such as highly-ordered and radiation-resistant single crystals. More importantly,
crystal packing constraints might hinder biologically relevant motions, and it simply cannot be
used to study irreversible reactions such as protein folding. These problems can be overcome by
applying time-resolved X-ray diffraction directly to protein solutions rather than protein single
crystals and then capturing transient molecular structures in one-dimension. To emphasize that
structural information can be obtained from the liquid phase, this time-resolved X-ray solution
scattering technique is named time-resolved X-ray liquidography in analogy to time-resolved Xray
crystallography where the structural information of reaction intermediates is obtained from
the crystalline phase. Time-resolved X-ray liquidography permits us to investigate the
tertiary/quaternary conformational changes of human hemoglobin triggered by laser induced
ligand photolysis in nearly physiological conditions. Data on optically induced tertiary relaxations
of myoglobin and refolding of cytochrome c are also reported to illustrate the wide applicability of
this technique. In addition the photocycle of PYP which has been extensively investigated with
time-resolved X-ray crystallography has been also studied with time-resolved X-ray liquidography
so that a direct comparison between liquidography and crystallography is possible. By providing
insights into the structural dynamics of proteins functioning in their natural environment, timeresolved
X-ray liquidography complements and extends results obtained with time-resolved
spectroscopy and X-ray crystallography.
References
[1] Ihee, H., Acc. Chem. Res., 42, 356-366 (2009).
[2] Cammarata, M. et al., Nature Methods, 5, 881 (2008)
[3] Lee, J. H. et al., J. Am. Chem. Soc., 130, 5834 (2008)
[4] Kong, Q. et al., Angew. Chem. Int. Ed., 47, 5550 (2008)
[5] Lee, J. H. et al., Angew. Chem. Int. Ed., 47, 1047 (2008)
[6] Kong, Q. et al., J. Am. Chem. Soc., 129, 13584 (2007)
[7] Kim, T. K. et al., Proc. Natl. Acad. Sci., 103, 9410 (2006)
[8] Lee, J. H. et al., J. Chem. Phys., 125, 174504 (2006)
[9] Ihee, H. et al., Science, 309, 1223 (2005)
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