Structural changes to proteins are absolutely critical for biological function, but how these structural changes come about is still somewhat of a mystery. One hypothesis suggests that proteins flex and stretch as a result of long-range vibrational motions mediated by interconnected webs of amino acid residues. The well-known phenomenon of allostery, where binding of a molecule or a mutation at a site distant to the active site has an effect on enzyme function, supports this hypothesis.

Researchers, however, have not had at their disposal a suitable approach to physically measure these long-range vibrations. “There's been a lot of skepticism about whether these motions actually exist,” says Andrea Markelz of the University at Buffalo, State University of New York. Although a handful of studies have reported X-ray scattering or Raman spectroscopy measurements attributed to long-range vibrational motions in proteins, Markelz notes, these measurements can be quite complicated to perform, and the preliminary results were never further explored with follow-up experiments.

Markelz has long suspected that the right technique would allow these elusive long-range protein vibrations to be detected. And now her team—including postdoc Gheorghe Acbas, graduate student Katherine Niessen and crystallography collaborator Edward Snell—has a method to do so.

Long-range vibrations in lysozyme are observed upon excitation with terahertz light. Image courtesy of A. Markelz and K. Niessen.

The team used a technique based on terahertz near-field microscopy, which allows vibrational measurements to be made under fully hydrated, room-temperature conditions. Terahertz-frequency light (found on the electromagnetic spectrum between infrared and microwave frequencies) induces different vibrational modes in a protein. “Just about everything in the cell absorbs this kind of light very well, so it's been hard to actually see these internal modes of the protein,” says Markelz. The key to singling out long-range vibrational modes over a sea of background noise, the team discovered, was to align the protein molecules via crystallization to create a strong signal. “You have to come up with some way to subtract out that background,” explains Markelz. “If every single protein is aligned, then we have a special direction where we're going to see a difference in the light versus any other direction.”

In this first proof-of-principle report, Markelz's team studied a protein called chicken egg white lysozyme. They identified long-range motions thought to be important for its antibacterial activity of cleaving cell-wall carbohydrates. The researchers' next task is to perform further experiments, such as using mutations or inhibitors to isolate particular vibrational modes, to get at the mechanism and biological function of these motions. “That's been our hope and dream all along,” says Markelz.

Markelz also hopes that traditional structural biology labs will be able to perform these sorts of dynamic measurements in the near future, once the technology becomes a bit more affordable. “The terahertz near-field microscope is definitely not something you buy off the shelf yet,” she notes. “It is a tabletop technique, but it does require an ultrafast laser.” However, these have become turnkey lasers and are rapidly dropping in price. It will certainly be interesting to watch whether the approach can be coupled with traditional structural biology tools to uncover new insights into protein function.