Many of the most interesting processes in nature are so fast, they can make “a blink of an eye” look like a millennium. Cellular proteins undergo elaborate transformations in as little as a picosecond – one millionth of one millionth of a second. That astonishing time scale presents an enormous challenge to scientists who would like to study the structure and behavior of those proteins. To catch the extremely fleeting moments of transition between different structural states of one such protein, a University of Chicago laboratory used a strategy straight out of science fiction: freezing time.
Xiaojing Yang, a senior research professional in the laboratory of Keith Moffat, wanted to look at a particular protein called a bacteriophytochrome, a red light photoreceptor from bacteria related to phytochromes in plants. Photoreceptors are found everywhere in nature, from plants to eyes, and are activated by light to change their shape and transform a light signal into a biological signal. Yang was interested in the contortions a photoreceptor makes when changing from the “dark” state to the “light” state, and chose a particular phytochrome from the bacterial species Pseudomonas aeruginosa.
Yang started with a method called X-ray crystallography, where proteins are maneuvered into crystal formation and then imaged at the atomic level using an X-ray beam. With traditional X-ray crystallography, Yang could determine the structure of the P. aeruginosa phytochrome in the dark state, before activation with the light signal – data she reported in a 2008 PNAS paper. But in order to see the transition between dark and light in finer detail, the researchers needed to develop a new trick.
“We applied an innovative application of crystallography called temperature-scanning cryocrystallography, where we use temperature to mimic time,” Yang said of the work, published this week in Nature. “So this is a new way of doing dynamic crystallography.”
Cryocrystallography, or “cryotrapping,” involves performing the method at a very low temperature to freeze the target molecule in a particular conformation. The temperature-scan aspect that Yang and colleagues added was to hold the crystal at an escalating series of temperatures (from -279° F to -135° F), each one moving the super-fast structural changes the slightest bit forward. The researchers could then do an X-ray scan at each temperature level to capture those normally-fleeting in-between stages.
“We shine the light at different temperatures, higher and higher,” Yang said. “The reaction progresses further as the temperature rises, and then you have a different mixture of reaction intermediates.”
“It’s a cunning way of slowing things down from picoseconds to minutes or even tens of minutes,” said Moffat, Louis Block Professor of Biochemistry & Molecular Biology at UChicago.
The experiments, using ten temperature levels in all, revealed three predominant intermediate states, called L1, L2, and L3. Transitions between states resemble a “molecular earthquake,” Moffat said, with the changes initially appearing at one corner of the light-sensitive chromophore of the phytochrome before spreading outwards until the entire structure is in flux. And as dramatic as the motion is, it’s only the initial steps of the phytochrome’s dance. Moffat uses the analogy of filming a sprint to describe the action they captured – the flash of light that starts the reaction is the starting gun – and says that they are so far only able to observe the first 15 meters of a 100 meter dash.
“We’re not in a position to observe the rest of the race,” Moffat said. “Yes, we would like to see the entire race going all the way to the finish, but we believe that the overall reaction from beginning to end involves quite a large molecular convulsion, which is probably not compatible with the crystals.”
With the more detailed information about how the phytochrome transitions from the dark to light state, Yang and colleagues could then run a functional test of how the receptor converts light to biological activity. In the natural setting, this phytochrome is linked to an enzyme called histidine kinase, a cellular switch that can launch a whole cascade of activity within the cell. When the researchers mutated a single amino acid that stabilizes two of the three intermediate states, they found that they blocked the ability of the bacteriophytochrome to activate histidine kinase – evidence of a link between structure and function.
While there is still much to learn about this particular phytochrome, the applications of that knowledge go beyond the realm of crystallography and microbiology. Because phytochromes are used by plants to determine core activities such as flowering time and germination, understanding the structure and function of the receptors might inform new bioengineering tools for agricultural use. Photoreceptors are also gaining popularity as a laboratory tool in the method known as optogenetics, where light-activated receptors are used to control processes like brain activity.
“If you understand how it works, then down the road we might be able to exploit that finding to make it work better, manipulate how it works, and transfer its properties to another protein that we engineer or design,” Moffat said. “So the system is of interest to a very wide range of people, all the way from agricultural scientists and plant physiologists on one end to biophysicists like us on the other.”
Yang, X., Ren, Z., Kuk, J., & Moffat, K. (2011). Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome Nature DOI: 10.1038/nature10506