Time-resolved serial synchrotron and serial femtosecond crystallography of heme proteins using photocaged nitric oxide
Peter Smyth, Sofia Jaho, Lewis J. Williams, Gabriel Karras, Ann Fitzpatrick, Amy J. Thompson,a Sinan Battah, Danny Axford, Sam Horrell, Marina Lučić, Kotone Ishihara, Machika Kataoka, Hiroaki Matsuura, Kanji Shimba, Kensuke Tono, Takehiko Tosha, Hiroshi Sugimoto, Shigeki Owada, Michael A. Hough, Jonathan A.R. Worrall, Robin L. Owen [1]
Molecular Tour
Time-resolved X-ray crystallography enables the capture of molecules movies which show how proteins in action, and is currently undergoing a renaissance due to the development of serial crystallography at synchrotron and XFEL beamlines. Crucial to such experiments are efficient and effective methods to uniformly initiate time-dependent processes within microcrystals, such as ligand binding, enzymatic reactions, or signalling. A common approach is the use of light to activate a reaction across the whole crystal at the same timepoint, however only a small proportion of protein reactions are light-activated. Diverse reactions can be made amenable to light-activation using photocaged substrates, which are molecules soaked into the crystal in advance, and release a reaction substrate upon laser activation. To collect high-quality time-resolved data, the reaction should be initiated in as many unit cells as possible within the crystal to provide structures with high occupancy of the activated states for easier interpretation, but without any damage caused by excessive light intensity, and without multiphoton excitation causing off-pathway reactions which do not reflect biological reality.
This work characterises photocage release of nitric oxide and binding of this ligand to two heme protein systems, cytochrome c′-beta and dye-decolourising peroxidase B, using a fixed-target sample delivery system. The differences between the structures of Methylococcus capsulatus cytochrome c′-beta as determined by traditional rotation crystallography at cryogenic temperatures, and via room-temperature serial crystallography, both in presence and absence of NO are described. The two bacterial proteins used here are ideal test cases, as the binding of NO results in the otherwise empty pockets on the distal side of the heme results in obvious changes in electron density. These structures have also allowed investigation of different methods for determining occupancy of the bound NO ligand, and how occupancy correlates to laser power.
Laser-activated SSX structures of DtpB. Omit electron density, contoured at 5σ maps for NO-bound heme site in chain C of DtpB, collected with different laser energies of
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With increasing laser power, the electron density maps support the presence of an NO molecule bound to the heme iron with increasing partial occupancy.
Laser parameters for photoactivation are systematically explored, and time-resolved structures over timescales ranging from 100 µs to 1.4 s using synchrotron and XFEL beamlines are described. The effective use of this photocage for time-resolved crystallography is demonstrated with laser powers as low as 0.19 µJ (~0.1 nJ∙µm-2), resulting in high occupancy NO-bound protein structures. Appropriate illumination conditions for such experiments are determined, and considerations for more general reaction initiation using photocages are discussed. An example is seen in the results of a high laser intensity illumination (1600 mJ, PDB: 9htc) McCP-β showing maps and chain A, for a and for an , contoured at 1σ and 5σ respectively. In this experiment, the fixed target chip was reversed in orientation compared to the other structures in this manuscript, and Mylar film was used to seal the chip, with the result that far less light reached the sample.
References
- ↑ Smyth P, Jaho S, Williams LJ, Karras G, Fitzpatrick A, Thompson AJ, Battah S, Axford D, Horrell S, Lučić M, Ishihara K, Kataoka M, Matsuura H, Shimba K, Tono K, Tosha T, Sugimoto H, Owada S, Hough MA, Worrall JAR, Owen RL. Time-resolved serial synchrotron and serial femtosecond crystallography of heme proteins using photocaged nitric oxide. IUCrJ. 2025 Sep 1;12(Pt 5):582-594. PMID:40843530 doi:10.1107/S2052252525006645
