Watching proteins move as they drive the chemical reactions that sustain life is one of the grand challenges of modern biology.
In recent years, X-ray free-electron lasers, or XFELs, have begun to meet that challenge, capturing ultrafast snapshots of molecules as they shift shape during a reaction — effectively creating molecular slow-motion movies.
But the technique comes at a cost: These experiments typically consume enormous amounts of precious protein samples, putting many studies out of reach.
Now, researchers at Arizona State University and their international colleagues, including scientists from the Consejo Superior de Investigaciones Científicas, have developed a device that cuts sample consumption by as much as 97% while still producing high-quality structural data. The device, called a microfluidic droplet injector, delivers protein crystals to an XFEL beam in tiny, precisely timed packets rather than as a continuous stream.
The technology could accelerate drug discovery by showing how medicines interact with their protein targets in real time and help engineers design better enzymes for industry and biotechnology. It may also enable deeper insights into disease, enable the study of rare proteins that are difficult to produce, and unlock the full potential of next-generation X-ray laser facilities without excessive sample waste.
"Seeing proteins react in real time is incredibly powerful, but the sample demands to unravel dynamic protein behavior with X-ray crystallography have been a major limitation," says Alexandra Ros , lead author of the new study. "Our droplet approach dramatically reduces that burden, which is exciting because many more labs can now ask dynamic questions that were previously too costly or impractical."
Professor Ros is a researcher with the Biodesign Center for Applied Structural Discovery and the School of Molecular Sciences at Arizona State University. She is joined by a team of ASU colleagues and international researchers, including collaborators from CSIC.
The new study appears in the current issue of the journal Communications Chemistry .
Freezing motion at the atomic scale
XFELs effectively freeze motion by firing ultrashort pulses lasting just femtoseconds — one quadrillionth of a second. In that tiny slice of time, light itself travels less than the width of a single strand of human hair.
In these experiments, proteins are first grown into microscopic crystals and then exposed to the XFEL pulses. This produces diffraction patterns that researchers use to reconstruct atomic-scale snapshots of the molecules in action.
In conventional XFEL experiments, protein crystals are sprayed continuously across the path of the X-ray beam. Only a small fraction of that material is actually hit by an X-ray pulse, meaning most of the protein goes to waste. The new system generates a train of microscopic droplets, each carrying a small amount of sample. These droplets are synchronized with the laser's pulse timing, so the sample arrives only when it is needed.
Working at the European X-ray free-electron laser, the researchers used time-resolved serial femtosecond crystallography, which can capture proteins as they change shape during reactions. With this powerful technique, they studied a human enzyme called NQO1, which plays an important role in cellular detoxification and protection against oxidative stress.
Big gains from tiny droplets
The researchers were able to capture early snapshots of how the enzyme begins interacting with its cofactor — a helper molecule called NADH — while using up to 97% less protein than traditional approaches. Understanding exactly how NADH binds and moves inside the enzyme could help scientists better understand how the enzyme functions and how it might be targeted in disease.
Although the biological findings are preliminary, the technological innovation is a significant advancement. By drastically reducing the amount of protein required, the method makes it feasible to study proteins that are rare, fragile or difficult to produce in large quantities. This opens the door to many experiments that were previously inaccessible.
The injector itself is built using high-resolution 3D printing and integrates tiny channels that mix solutions and form droplets on demand. The design is compatible with the extremely rapid pulse structure of modern XFEL facilities, allowing researchers to fully exploit the capabilities of these billion-dollar instruments without wasting valuable samples.
It could also complement emerging efforts to make X-ray laser technology more compact and accessible, including the compact X-ray free-electron laser currently under development at ASU.
