Structural biologists capture detailed image of gene regulator’s fleeting form

Using an ultrafast, high-intensity radiation source called an X-ray free-electron laser (XFEL), scientists have captured an atomic-level picture of an RNA structure called a riboswitch as it reorganizes itself to regulate protein production. The structure has never been seen before, and likely exists for only milliseconds after the riboswitch first encounters its activating molecule.

“We showed that structural changes in biochemical reactions or interactions between molecules can now be captured in vivid detail in real time with the use of state-of-the-art X-ray lasers,” says Petra Fromme, director, Biodesign Center for Applied Structural Discovery and professor at the School of Molecular Sciences at ASU.

The study opens the possibility of applying bacterial switching mechanisms to the fight against deadly disease.

The research, appearing in the advanced online edition of the journal Nature, represents the first time researchers have visualized the molecular structure of a biomolecule in such a transient form directly with an XFEL in real time, through a process known as ligand diffusion. The breakthrough suggests that researchers can now capture snapshots of molecular structures quickly enough to see even the fastest biological processes unfold.

The work was carried out by an international team, including researchers from Arizona State University, who worked with their colleagues at the Center for Cancer Research at the National Cancer Institute. The team also included scientists from the Center for Free-Electron Laser Science in Germany, the Linac Coherent Light Source at Stanford University, and Johns Hopkins University. The study was led by Yun-Xing Wang, a Senior Investigator in CCR’s Structural Biophysics Laboratory.

Wang’s lab has been studying riboswitches, structures at the ends of some messenger RNA molecules that reconfigure themselves in the presence of a ligand to alter gene expression. From biophysical experiments, the team had reasoned that when the adenine riboswitch binds to its ligand, its structure changes twice, passing through a short-lived configuration before adopting the ligand-bound form that others had observed. 

The intermediate state is so fleeting that its structure could not have been determined using conventional tools. While transient states have been seen before using large crystals or in photoactivated systems, combining XFEL with micro- and nanocrystals gave the team an opportunity to capture a ligand-triggered intermediate state during its brief existence.

“Almost all proteins, RNAs, and DNAs interact with ligands or substrates and undergo certain conformation changes when they react. The ability to visualize these changes is critical to understanding how biomacromolecules perform their functions,” says Wang. Using an innovative new method, researchers were able to directly visualize the structures and changes in real time. Such a capability has not been possible in the past 60-plus years of the crystallography history.

The researchers studied a riboswitch from the bacterium Vibrio vulnificus, a close relative of the cholera germ. It can cause infections that are especially hard to treat and often fatal. The switch is activated by a binding molecule, known as a “ligand,” in this case adenine. Upon activation the riboswitch changes its form. In the future, targeting the genetic switches in bacteria could offer a powerful weapon in the fight against disease.

The tool of choice for these detailed protein investigations is the XFEL, which can generate extremely bright X-ray light in pulses measured in femtoseconds, or quadrillionths of a second. The pulses last less time than it takes light to travel the width of a human hair, and an infinitely small fraction of the duration of a chemical reaction.

“With an exposure time of about 200 femtoseconds, this is surely the fastest camera in the world. And the process we capture is closely similar to the first event in human vision, when a light photon strikes the back of your eye,” said ASU physicist John Spence, a contributor to the Nature paper.

Crystals scatter X-rays in characteristic ways, and from the resulting diffraction pattern, the structure of the crystal can be calculated down to the atomic level, yielding the detailed structure of the aptamer in this case.

With the conformational change of the aptamers, the entire crystal structure and symmetry changes. Such experiments require very small crystals, as bigger crystals would simply fall apart due to the internal strain resulting from this rearrangement.

In the current study, tiny crystals of RNA were prepared — so small that a ligand could quickly and evenly diffuse through them. XFEL’s exceptionally bright light was essential for studying crystals of this size, and allowed the team to capture the riboswitch in its intermediate state.

The tiny aptamer crystals were injected into the ultra bright beam of the X-ray laser LCLS at the SLAC National Accelerator Laboratory in California. The technique involves a novel viscous medium into which the aptamer crystals were embedded and an injector system developed at ASU. These method developments were crucial for the study as they reduced the sample amounts required for the data collection by more than a factor of 100.

Surprisingly, the team also discovered that after ligand binding, the RNA molecules packed into the their micro- and nanocrystals rearranged themselves into a new crystal form, presumably due to the large conformation changes that transformed the intermediate state into the riboswitch’s final ligand-bound form, much like synchronized swimmers, (see accompanying video).  

The observation suggests that with crystals of this size, XFEL could be used to capture the structures that form during biochemical reactions from beginning to end.

By using XFEL to collect serial images of macromolecules in action, researchers can essentially create movies of biological processes with atomic-level detail. The new approach moves research from static, 3-D space to four dimensions, by adding the time element.  

The team of ASU researchers has worked with other scientists on the sample characterization, developed the embedding of the aptamer crystals in viscous media and delivered them to the XFEL beam in a viscous jet, and worked on the data collection and evaluation at LCLS. The group is supported by the BioXFEL Science and Technology Center funded by the National Science foundation, as well as grant funding from the National Institute of Health.

The ASU research team is highly interdisciplinary and involves researchers from the Biodesign Center for Applied Structural Discovery, the School of Molecular Sciences and the Department of Physics. The ASU team includes the graduate students Chelsie E. Conrad, C. Li and  Jesse D. Coe, the researcher and PostDoc Garret Nelson as well as the faculty Uwe Weierstall, Petra Fromme, Nadia A. Zatsepin and John C. H. Spence. Chelsie Conrad, Ph.D. student in the School of Molecular Sciences, who has developed the viscous crystal carrier, will graduate in December with a Ph.D. in Biochemistry and will join the lab of Dr. Wang at the Center for Cancer Research at the National Cancer Institute as a PostDoc, continuing this collaboration between ASU and the CCR.