Volume 94 Issue 46 | p. 7 | News of The Week
Issue Date: November 17, 2016

X-ray technique captures fast ligand-binding dynamics

Rapid mixing device combined with tiny crystals enables researchers to watch riboswitch in action
Department: Science & Technology
News Channels: Biological SCENE, Analytical SCENE
Keywords: reaction mechanisms, serial femtosecond X-ray crystallograpy (SFX), X-ray free-electron laser, enzyme mechanisms, riboswitch
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Femtosecond crystal structures of a riboswitch binding pocket show two unbound forms (blue and cyan), an intermediate state (yellow) in which a ligand (green) has just bound, and a final bound state (magenta) in which the riboswitch is fully activated.
Credit: National Cancer Institute
Femtosecond crystal structures of riboswitch binding site show two apo (unbound) forms, intermediate state in which a ligand has just bound, and final bound state in which the riboswitch is fully activated.
 
Femtosecond crystal structures of a riboswitch binding pocket show two unbound forms (blue and cyan), an intermediate state (yellow) in which a ligand (green) has just bound, and a final bound state (magenta) in which the riboswitch is fully activated.
Credit: National Cancer Institute

In the past, researchers have used X-ray free-electron lasers (XFELs) to take movies—rapid-fire crystal structure snapshots—of biomolecular processes. These processes had to be activated by light rather than ligand binding, a more common occurrence in nature. Because of typically slow ligand diffusion times and other timing difficulties, XFEL-based serial femtosecond crystallography (SFX) has not been used successfully to analyze binding-initiated processes.

Now, researchers have overcome these limitations by using a recently developed device to analyze crystals so tiny that ligands diffuse into them nearly instantaneously. A collaborative team led by structural biophysicist Yun-Xing Wang of the National Cancer Institute, in Frederick, Md., used the new SFX technique to reveal previously inaccessible details about how a riboswitch works (Nature 2016, DOI: 10.1038/nature20599).

Ligand binding causes riboswitches, regulatory segments of mRNA, to undergo conformational changes that turn gene transcription on or off. The new SFX technique “has a unique ability to capture key details of RNA switching in action,” says riboswitch expert Ronald Breaker of Yale University. Riboswitches are found primarily in bacteria and are therefore potential antibiotic targets. So SFX might help assess shape changes in riboswitches induced by antibacterial drug prospects, Breaker adds.

The Wang group carried out the new study by using approximately 1-µm-diameter riboswitch crystals—so small that a ligand was able to diffuse into a crystal and activate all its riboswitch molecules nearly instantaneously. A T-junction device rapidly mixes a ligand in one of its arms with tiny riboswitch crystals in the other. After a time delay, a nozzle shoots the mixture through an XFEL beam. Structures determined from diffraction patterns produced by the XFEL beam at different delay times reveal riboswitch conformation changes. Such shape changes would strain and break most regular-size biomolecular crystals, but the crystals used in the study are so small that they remain intact.

The SFX study, conducted with an XFEL at the SLAC National Accelerator Laboratory, produced crystal structures of two “apo,” or unbound, states; an intermediate bound state; and a final bound state of the riboswitch. Although the final state had been structurally analyzed before, SFX revealed new details about riboswitch activation.

SFX pioneer Janos Hajdu of Uppsala University says the technique might also be used to determine enzyme mechanisms initiated by substrate binding. In fact, “any mixing approach that causes a biomolecule to change structure could be a great application of this technology down the road,” adds RNA specialist Nils Walter of the University of Michigan.


CORRECTION: On Nov. 18, 2016, this story was updated to correct the name of SLAC National Accelerator Laboratory.

 
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ISSN 0009-2347
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