In a groundbreaking experiment using the world’s fastest camera, a team of scientists led by the University of Wisconsin-Milwaukee documented the fundamental processes of a chemical reaction as they occurred in real time. This means seeing how proteins, the building blocks of life, work in a few quadrillionths of a second.
In a paper, published May 5, 2015, in the journal Science, the researchers describe how they acquired images of the effect of light on a tiny crystallized protein.
Light absorption by proteins is the primary event of processes such as vision and photosynthesis that are fundamental to many life forms, including humans as well as plants and bacteria. The researchers focused on photoactive yellow protein, a light-absorbing component found in certain bacteria. The basic chemical process they observed, known as isomerization, also occurs when the retina in the human eye responds to light.
Understanding how these complex molecules do their job depends on knowing the spatial arrangement of atoms and how their structure changes as they interact with light. Until now, no effective method for detailed observation of molecular movement in such detail was available. The Science paper describes an experimental first.
“This puts us dramatically closer to understanding the chemistry necessary for all life,” said Marius Schmidt, PhD, professor of physics at UW-Milwaukee and the leader of the team. “Discovering the step-by-step process of how proteins function is necessary not only to inform treatment of disease, but also to explore the grand questions of biology.”
“Light drives much of biology and this novel experiment is a pinnacle in understanding how living systems respond to light”, said University of Chicago professor and team member Keith Moffat, who pioneered this experimental approach and, with his Chicago colleagues, developed it over 25 years.
The data were collected using the Linac Coherent Light Source X-ray free electron laser, or XFEL, at the SLAC National Accelerator Laboratory – operated by Stanford University for the U.S. Department of Energy Office of Science.
At the speed of life
“Biology happens at short time spans,” explained co-author Jason Tenboer, post-doctoral researcher in Schmidt’s lab. The Linac Coherent Light Source supplied the team with camera frames each lasting about 150 femtoseconds, about 1,000 times faster than any seen in an X-ray experiment before and rapid enough to allow the imaging of the fastest reactions.
Unveiling the atomic-scale changes in protein molecules as they go about their tasks is important because these changes in structure determine their function. In 2014, Schmidt and colleagues were the first to document changes in a protein molecule over a larger increment of time.
The scientists mapped the atoms in motion in the photoactive yellow protein as the chemical bonds of a central dye molecule – which is buried within the protein and makes it yellow – rearranged. They documented, for the first time, the structure of the yellow dye within the protein in an electronically excited state.
For the past 60 years, the only way to examine proteins in three dimensions was with X-ray crystallography. This process shoots X-rays at crystallized proteins. These proteins 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. But it is a still snapshot, a single point in time when nothing is moving.
To capture protein molecules in action, scientists need both an optical laser and an X-ray laser with split-second pulses, which is what the XFEL provides.
Next, the researchers hope to collect femtosecond details over a bigger range of time to create a slow-motion “movie.” This could ultimately allow scientists to intervene in the process of protein functions by using light.
“We’re interested in the mechanism of the chemical reaction, with the goal of controlling and steering it in a certain direction with light,” Schmidt said. “We can shape laser pulses for that purpose. We will discover how the molecules march in synchronicity during such processes.”
Forty-four researchers were involved in this study. In addition to those from UW-Milwaukee, Chicago and Stanford, members of the scientific team came from Arizona State University; Lawrence Livermore National Laboratory; University of Hamburg; State University of New York, Buffalo; University of Jyvaskyla; Max Planck Institute for Structure and Dynamics of Matter; and Imperial College, London.
Moffat and University of Chicago researchers pioneered time-resolved X-ray crystallography. Beginning in the 1990s, using the National Synchrotron Light Source (Brookhaven National Laboratory), the European Synchrotron Radiation Facility (Grenoble, France), and the BioCARS facility at the Advanced Photon Source (Argonne National Laboratory), they reduced time resolution from milliseconds to nanoseconds to 100 picoseconds. Now, thanks to the Linac Coherent Light Source at Stanford, the team has cut time resolution to 150 femtoseconds, the time scale on which fundamental chemical reactions take place. Moffat and colleagues were the first to conduct time-resolved crystallography and developed photoactive yellow protein as a paradigm for light-dependent signal transduction in biology.