Biology: Time-Resolved Crystallography

Studies of macromolecules by the X-ray diffraction technique over the last few decades have provided unprecedented insight into their function by providing the average, static 3-D structures of macromolecules. However, to fully elucidate how these molecules perform their function, one must watch them in action, along a reaction path that often involves short-lived intermediates. Time-resolved crystallography is a unique tool for achieving this goal because it provides direct, detailed and global structural information as molecules in the crystal undergo structural changes.

In time-resolved experiments, a reaction is rapidly triggered in molecules in the crystal, and short X-ray pulses are used to probe structural changes at various time delays following the start of the reaction. Time resolution of 100ps, matching the duration of a single X-ray pulse at the synchrotron source, has been achieved (Schotte et al., 2003). Reaction triggering is a crucial part of the experiment. The fastest method for triggering a reaction in the crystal involves use of ultra-short (fs to ns) laser pulses. This method is clearly suitable for inherently photosensitive molecules that undergo structural changes upon the absorption of light by an embedded chromophore. Alternatively, for proteins that are not inherently photosensitive, photo-triggering can be accomplished by using caged compounds.

Time-resolved experiments that require sub-second time resolution utilize the polychromatic, Laue X-ray diffraction technique, where the crystal is kept stationary during the X-ray exposure. A comprehensive review of the present, mature state of the Laue technique as well as examples of its application to static and time-resolved studies can be found in Ren et al., 1999. BioCARS staff scientists played an essential role in the development of all aspects of time-resolved crystallography. This technique has successfully advanced to a mature stage with the use of high-flux third-generation synchrotron sources, demonstrated ability to detect small structural changes even at relatively low levels of reaction initiation of 15-40% (Srajer et al., 1996; Ihee et al. 2005; Rajagopal et al., 2005) and with significant advances in processing and analysis of time-resolved Laue crystallographic data (Srajer et al., 2001; Ren et al., 2001; Schmidt et al., 2003; Rajagopal et al., 2004). A particularly important development is the application of the Singular Value Decomposition (SVD) method to the analysis of time-resolved crystallographic data (Ihee et al. 2005, Schmidt et al., 2003; Rajagopal et al., 2004; Rajagopal et al., 2005).

For the review of time-resolved crystallography in practice see:

V. Šrajer: Time-resolved Macromolecular Crystallography in Practice at BioCARS, Advanced Photon Source: From Data Collection to Structures of Intermediates
Published in “The Future of Dynamic Structural Science, NATO Science for Peace and Security Series A: Chemistry and Biology”
(J.A.K. Howard, et al, Eds.), Springer Science+Business Media Dordrecht, Chapter 17, pp. 237-251 (2014)
(The final publication is available here)



Science Highlight

Jung, Y. O., Lee, J. H., Kim, J., Schmidt, M., Moffat, K., Šrajer, V., and Ihee, H. (2013) Volume-conserving trans–cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography. Nature Chemistry 5, 212–220.

Trans-to-cis isomerization, the key reaction in photoactive proteins, usually cannot occur through the standard one-bond-flip mechanism. Owing to spatial constraints imposed by a protein environment, isomerization probably proceeds through a volume-conserving mechanism in which highly choreographed atomic motions are expected, the details of which have not yet been observed directly. Here we employ time-resolved X-ray crystallography to visualize structurally the isomerization of the p-coumaric acid chromophore in photoactive yellow protein with a time resolution of 100 ps and a spatial resolution of 1.6 Å. The structure of the earliest intermediate (IT) resembles a highly strained transition state in which the torsion angle is located halfway between the trans- and cis-isomers. The reaction trajectory of IT bifurcates into two structurally distinct cis intermediates via hula-twist and bicycle-pedal pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue through E46Q mutation, which switches off the bicycle-pedal pathway.





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