For a protein to carry out its job—whether it be replicating DNA, metabolizing fuel, transporting biomolecules, or sending cell signals—its amino acids have to move in certain ways. The patterns of these internal motions aren’t always well understood because the tools available to study them are limited.
A new technique, electric field-stimulated X-ray crystallography (EF-X), combines electric pulses with time-resolved X-ray crystallography to provide more comprehensive views of the ways proteins work. Electrical charges and dipoles are present in all proteins, and external electric fields can exert forces on them, causing atoms to move. In EF-X, developed by a research team led by Rama Ranganathan of the University of Texas Southwestern Medical Center, electric field pulses activate conformational changes of amino acids throughout protein molecules in crystals, and fast X-ray pulses then visualize those motions (Nature 2016, DOI: 10.1038/nature20571).
EF-X has potential advantages over other techniques for seeing protein motions. Nuclear magnetic resonance spectroscopy can analyze motions of specific nuclei in proteins, but the movements must be inferred indirectly from chemical shifts and other NMR parameters. EF-X can visualize the motions directly. Time-resolved X-ray crystallography generally uses light to initiate biological processes, but that works only in proteins with chromophores. Light also has more energy than is required for typical conformational changes. In contrast, EF-X can initiate subtle movements in proteins without light. And force spectroscopy does not have the spatial resolution that EF-X has to see detailed internal atomic-scale protein motions.
Ranganathan and coworkers used EF-X to visualize motions in a human PDZ domain, a common motif in cell signaling proteins. Electric pulses induced conformational changes throughout the domain, driving it into low-level excited states that the researchers visualized. Structures of excited-state species aren’t normally accessible with crystallography.
The scientists observed patterns of induced motions throughout the domain, and those patterns corresponded to conformational changes that occur naturally when ligands bind to PDZ domains. The work thus “lays the foundation for comprehensive experimental study of the mechanical basis of protein function,” the researchers note in their paper.
EF-X could be more generally applicable than existing techniques for initiating fast structural changes and assessing their functional consequences, says Keith Moffat, director of the University of Chicago’s BioCARS facility, where Ranganathan and coworkers did the study’s X-ray work. But Moffat cautions that some electrically induced changes may turn out to be not functionally meaningful.
“This is an experiment I never thought would work, so it’s quite amazing that it does,” says protein dynamics expert Steven G. Boxer of Stanford University. “I’m still not convinced EF-X will be broadly applicable” in all cases, especially when proteins are undergoing large structural changes, Boxer adds. For example, how a protein in a crystal happens to be oriented relative to the applied electric field direction may affect the results of EF-X experiments, he says. “But it’s cool to see this first step, and I would be happy to be wrong.”