Living organisms operate like factories, producing continuous chemical reactions, with proteins serving as the laborers. Every process that sustains life depends on these complex molecules.

Discovering how proteins do their jobs holds incredible potential for improving lives. But unraveling their functions depends on seeing how the atoms are arranged and how they change during reactions. Until recently, only a fraction of the world’s proteins could be observed with current technology.

Now, two UW-Milwaukee scientists have helped usher in a new age of unmasking proteins, using a tool that images extremely small objects with unprecedented speed and clarity. In a groundbreaking experiment, UW-Milwaukee physicist Marius Schmidt and his doctoral student, Jason Tenboer, became the first to witness a protein changing in real time with the X-Ray Free Electron Laser, or XFEL. The feat has opened the door to resolving what Schmidt calls “some of the grand challenges of biology – understanding the molecular basis of life.”

The nano-moment of truth

A protein is far smaller than a single cell. About 3,000 different proteins operate in the common bacterium E. coli, for example.

For the past 60 years, the only way to examine proteins in 3-D was with X-ray crystallography. The process shoots X-rays at crystallized proteins, which diffract light and create patterns of dots the way shaking a paintbrush sprays drops on a wall.

The pattern provides a fingerprint for that protein. The millions of data points can be mathematically reconstructed to form a 3-D image of the protein’s molecular structure at a single point in time – a still snapshot.

But this method works with less than 20 percent of proteins – only those that crystallize in a small enough size – and it can’t capture them in action. For that, scientists need a laser with split-second X-ray pulses.

They found the tool they needed at the Stanford Linear Accelerator Center in Menlo Park, California.

With a light source that’s a billion times brighter than any other equipment, the XFEL transforms X-ray crystallography into a kind of high-definition video, providing ultraslow-motion of extremely rapid events.

Schmidt and Tenboer spent about six months growing protein crystals at UW-Milwaukee before heading west. Deciding it was too risky to fly the delicate cargo, they carefully packed the specimens into a climate-controlled van for the long drive to California.

In a quadrillionth of a second

Once there, they worked with a team of 34 other scientists from nine institutions to put the XFEL to the test. The group included four additional UW-Milwaukee researchers: Abbas Ourmazd, Peter Schwander, Chris Kupitz and Jennifer Scales.

The team induced a chemical reaction in a protein crystal the size of a bacterium and then bombarded it with X-rays. With data generated by the signature “sprays” of photons, they created high-resolution “maps” documenting electrons in the protein molecules shifting during the reaction.

“Biology happens at inconceivably short time spans,” Tenboer said. “So the XFEL allows us to do time-resolved studies of proteins in action down to the femtosecond time scale – that’s 10-15 of a second.”

Since their first experiment, Schmidt and Tenboer have conducted a second experiment, making them the first to observe a chemical reaction unfold among multiple proteins working together.

“This study shows that the molecular details of life’s chemistry can be followed using X-ray laser nano-crystallography,” Schmidt said, “which puts some of biology’s most sought-after goals within reach.”

Schmidt and Tenboer’s paper was published last year in the journal Science. The UW-Milwaukee physicists named here are members of a prestigious $25 million BioXFEL Science and Technology Center funded by the National Science Foundation.

Researchers from UW-Milwaukee and SLAC were joined by researchers from Arizona State University; Lawrence Livermore National Laboratory; University of Hamburg and DESY in Hamburg, Germany; State University of New York, Buffalo; University of Chicago; and Imperial College in London. The work was supported by the National Science Foundation, National Institutes of Health and Lawrence Livermore National Laboratory.