The structure of a crystallized protein (center) changes as it reacts to light (left). The X-ray laser (bottom) hits the crystal, diffracting photons, allowing scientists to recreate a 3-D snapshot of the protein.
Photoisomerization—a biological response to light—is a photon-driven change in a protein’s molecular structure. This reaction happens almost instantaneously, making it impossible to observe in real-time. Now, an international team of researchers from 11 different institutions reports a new method they say can capture this split-second structural transition using X-ray pulses emitted by a free electron laser (FEL) source (Science, doi: 10.1126/science.aad5081). “Light drives much of biology, says team member Keith Moffat, University of Chicago, USA, “and this novel experiment is a pinnacle in understanding how living systems respond to light.”
Ultrafast pulses for ultrafast timescales
The researchers, led by Marius Schmidt, University of Wisconsin, Milwaukee, USA, investigated the structural dynamics of the chromophore in photoactive yellow protein (PYP). Absorption of light causes the PYP molecule to change from its original “trans” form to its “cis” form. Knowing the step-by-step process of how PYP’s structure changes will help scientists better understand how it behaves—in other words, its structure determines its function. This transition, called trans-to-cis photoisomerization, happens almost instantaneously—too fast for standard X-ray crystallography to capture.
The U.S. Department of Energy’s SLAC National Accelerator Laboratory’s FEL laser (called the Linac Coherent Light Source, LCLS) emits hard X-ray pulses on the femtosecond timescale, fast enough to capture PYP’s trans-to-cis photoisomerization. However, the researchers needed to replace SLAC’s optical laser with one that could pulse just as fast as LCLS and excite the photoactive protein. By matching the pulse speeds of both lasers, the researchers say they captured the structural dynamics of the reaction on the same femtosecond timescale as the pulses.
Real-time serial femtosecond crystallography
In order to record PYP trans-to-cis photoisomerization, the researchers prepared microcrystals of the protein. The crystalized PYP molecules were placed in a test chamber and each crystal was hit with pulses from the optical laser. Next, a hard X-ray pulse from LCLS would capture the “image” of the crystalized protein’s structure change in response to the optical laser pulse. Schmidt and his colleagues varied the time between the optical and X-ray pulses from 100 femtoseconds to 3 picoseconds to see how the protein shape changed over time.
The researchers say that they were able to capture atomic motions as fast as 100 quadrillionths of a second—1,000 times faster than their previous efforts—capturing the structural change of the PYP molecule in an electronically excited state. Based on their data, the trans-to-cis photoisomerization happens about 550 femtoseconds after the molecule absorbs light, which corresponds with theoretical calculations.
The researchers say their method could inform other studies on light-driven structural dynamics, like how pigments in the eye respond to light; how photosynthetic organisms turn light into chemical energy; and how atomic structures respond to light pulses of different shapes and durations.