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Study sheds X-ray light on mysteries of photosynthesis

Study gives most detailed picture to date of nature at work at tiniest scale.
High-speed photography at speed of femtoseconds (quadrillionth of a second).
May 5, 2016

ASU researchers part of international team whose work at minute scale could lead to big innovations in energy, health and more

High-speed photography can capture a horse’s gallop, a falling star or even a speeding bullet. But such methods would be far too slow to record the elusive movements of protein molecules as they undergo transitions from one form to another — a process known as isomerization.

In new research appearing in the journal Science, an international team of researchers used brilliant bursts of X-ray light to capture the movements of a photosensitive protein — one that enables a broad range of life forms to convert light into energy.

The research represents the most detailed picture to date of nature at work at the tiniest scale. Results of the new study could pave the way for innovations ranging from renewable energy to a new generation of bio-inspired sensing devices. Similar investigations of protein structure using pioneering techniques in high-speed X-ray crystallography could also usher in a suite of new drugs, targeting a range of human diseases.

Marius Schmidt from the University of Wisconsin-Milwaukee and his colleagues led the research effort. The group was joined by researchers from the Stanford Linear Accelerator (SLAC), where the experiments were performed in collaboration with multiple institutions from around the world.

Researchers at Arizona State University’s Biodesign Institute contributed to the data analysis of the experiments, which tracked the subtle contortions of photosensitive yellow protein (PYP) over an astonishingly short time frame, running from 100 femtosecondsA femtosecond is one quadrillionth of a second. A picosecond is one trillionth of a second. to 3 picoseconds.

“Once the PYP protein absorbs a photon of light, it changes its shape from an initial configuration known as the trans form to a new shape known as cis,” said Petra Fromme, director of the Biodesign Center for Applied Structural Discovery. “The trans to cis transition occurs in such a unbelievably brief time span that nobody had been able to see the important details of this process — until our discovery.”

To give a sense of the brevity of the X-ray laser pulses illuminating the protein, there are as many femtoseconds in one second as there are seconds in 32 million years.

Light moves

Many organisms have developed sophisticated means to detect and respond to photons of light, through changes in protein structure initiated by photon absorption. The star attraction for the XFEL (X-ray Free-Electron Laser) study is PYP — a particular protein found in purple bacteria, used for sensing and responding to blue light. PYP works much like photosensors in the human eye, though the chemicals involved are different.

Photoreceptors of various kinds occur in plants, algae and fungi as well as in bacteria. Among their functions, photoreceptors help organisms reorient themselves toward or away from light, which is useful for protection against damaging high-energy light as well for maximizing  sunlight exposure to grow using photosynthesis.

Scientists hope to learn more about the dynamics of biological substances, including proteins like PYP, but many have been difficult to study with conventional X-ray crystallography. The process involves crystalizing a sample of interest, then striking the crystals with a stream of X-rays, producing diffraction patterns, which are then assembled into a working structure of the protein in question. For many proteins however, growing crystals of sufficient size is a severe challenge.

Sea change

A revolution in protein imaging is now underway thanks to XFELs, which are devices that produce intense X-rays suitable for use with very tiny crystals.

XFELs yield their riches in the form of diffraction patterns before the X-ray pulse obliterates them. The method is sometimes referred to as diffract-and-destroy. Shorter X-ray pulses with greater intensity offer improved structural information.

The extremely short durations of the brilliant X-ray pulses, produced at the Stanford Linear Accelerator’s XFEL, are necessary to capture the fleeting oscillations of the PYP photoreceptor, but also provide other critical advantages, allowing tiny microcrystals to be used, rather than much larger crystals common to traditional X-ray crystallography. 

Unlike earlier methods requiring crystalized samples to be kept at a chilly minus-173 degrees Celsius, the photosensitive protein under study was maintained at room temperature, more closely resembling its natural environment.

The powerful, femtosecond X-ray pulses produced by Stanford’s Linac Coherent Light Source (LCLS) are a billion times more brilliant than X-rays produced by synchrotrons. These highly energetic bursts actually destroy the crystals under observation, but the pulses are so short that diffraction images used to determine structure can be gathered before the sample is vaporized.

Essentially, the femtosecond pulses outrun radiation damage. By assembling multiple images, researchers can make detailed movies of biological molecules in motion.

“With an exposure time of about 200 femtoseconds, this is surely the fastest camera in the world. And the process we capture is closely similar to the first event in human vision, when a light photon strikes the back of your eye,” said ASU physicist John Spence, a contributor to the new study.

Capturing the movements of life

In previous studies, the researchers showed that serial femtosecond crystallography could successfully monitor the PYP structure in nanosecond-to-microsecond time scales (a thousandth to a millionth of a second). The new technique was used to reveal the fine details of cis to trans isomerization of the PYP protein in real time; in particular, its movements during the critical transition from 100 to 3000 femtoseconds.

In the current study, researchers prepared crystallized samples of PYP just 2 millionths of a meter in length, exposing each to blue laser light before injecting them into the LCLS X-ray beam. The trans to cis isomerization was found to occur roughly 500 femtoseconds after the protein’s absorption of light, with the initial transition taking place after roughly 250 femtoseconds.

In the dark, the PYP chromophore or antenna is in the trans condition and the protein is tightly folded. Absorption of a blue photon turns the chromophore yellow and induces isomerization to the cis state, in which the protein partly unfolds. The process is reversible, with the protein reverting to its folded trans state when blue light is removed.

As the authors note, the transition is marked by physical extremes, with the light-sensitive chromophore accelerating by 2 x 10 to the 15th m/s2, with a final velocity reaching 500 m/s (over 1100 mph).

Detailed studies of PYP dynamics open the door to explorations of a dizzying array of more complex proteins vital to life processes, observed at ultrafast timescales.

ASU researchers on the project are all members of the Biodesign Center for Applied Structural Discovery with appointments in the School of Molecular Sciences or Department of Physics, including faculty Petra Fromme, John Spence, Raimund Fromme, Nadia Zatsepin, Uwe Weierstall and graduate students Shibom Basu, Chelsie Conrad, Shatabdi Roy-Chowdhury, Jesse Coe, Gihan Ketawala and Ganesh Subramanian, Daniel James.

Top image: This illustration depicts an experiment at the Stanford Linear Accelerator (SLAC) that revealed how a protein from photosynthetic bacteria changes shape in response to light in less than a trillionth of a second. Samples of the crystallized protein (right), called photoactive yellow protein, or PYP, were struck by an optical laser beam (blue light coming from left) that triggers shape changes in the protein. These were then probed with a powerful X-ray beam (fiery beam from bottom left) from SLAC’s Linac Coherent Light Source. Image by SLAC National Accelerator Laboratory

Richard Harth

Science writer , Biodesign Institute at ASU

480-727-0378

 
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For the love of family

May 5, 2016

With his daughter as inspiration and his mother's support, Yuri Lechuga-Robles turns his life around, discovers love of landscape architecture

Editor's note: This is part of a series of profiles for spring 2016 commencement. See the rest here.

Yuri Lechuga-Robles rebuilt his life the minute his daughter was born. In those first moments she came into the world, he knew it was time to lay the groundwork for a new future.

Arizona State University simply provided the means.

Born in Cuernavaca, Mexico, Lechuga-Robles moved around quite a bit in his youth. He spent part of his childhood in El Paso; then, when his mother remarried, his family settled down in Mesa when he was around 11. Lechuga-Robles found his passion in a high-school drafting class, eventually attending High Tech Institute. Despite landing a job with a firm and living what he thought was his dream, he lost his way, struggling with drugs and alcohol.

Then he had his daughter, AnnaMariah Robles-Lantigua (pictured above with Lechuga-Robles and his mom, Maria Budzinski), and pruned away all the negative forces from his life. He said that after her birth, he took the first steps to “get back to the values that were taught to me.”

Charging forward with a renewed outlook, and a mission to “finish something,” Lechuga-Robles got busy and plugged in.

He went back to school at Mesa Community College (MCC), where he first learned about landscape architecture, before transferring to ASU. He joined the Student Chapter of the American Society of Landscape Architects and the Phi Theta Kappa honors society as a dedicated member of both groups. This found Lechuga-Robles organizing a book fundraiser for the Boys & Girls Club to reestablish the club library lost in a fire many years ago.

Eventually his dedication to the community was recognized by the Arizona Department of Transportation, and he was awarded the John McGee Intern Scholarship — the first landscape architecture student to receive this award.

Days away from being handed a bachelor’s of landscape architecture through the Design School in the Herberger Institute for Design and the Arts, Lechuga-Robles — who will be 10 years sober in December — is reflective about the past and looking forward to a bright future.

Question: What was your “aha” moment, when you realized you wanted to study the field you majored in?

Answer: At MCC and talking with LeRoy Brady (the landscape architect of the rose garden at MCC) and one of my other professors — through one of the assignments we had in one of those classes was to design a new building for architecture if MCC acquired new land. That’s where I saw I was really interested in outside spaces. … When I came to ASU I still had my mind kind of set on architecture. Then I learned about the 3+ program and I thought, “Why would I want to go six years to get one license when I could go seven and get two licenses: architecture AND landscape architecture?” When it came time to decide which way I wanted to go, I just went that way. And now I don’t want to do the other. There’s enough in landscape architecture for me.

One of the most significant projects I worked on at ASU was the 30-year with Kristian Kelley (LDE 362/590: Landscape Architecture II). We had to look at a space in downtown Phoenix kind of in the south central area. It deals a lot with the culture, a lot of low-income residences. And just on the other side of the tracks there’s these office buildings and the ballpark. It’s a big contrast, and I really enjoyed connecting the two and still keeping the character of the neighborhood and bringing in some of their cultural values and things that they’re proud of.

That was my favorite project. It gave me a sense of focus and direction for what I wanted to do with landscape architecture, because there’s so many avenues you can go on with that degree. I’m interested in urban infill and urban design, projects that deal with social justice, thinking about current issues. I see people flying off the handle, and it seems to me they don’t have a way to connect with other people, a way to release. I draw from my experience dealing with drugs and alcohol. I see maybe there’s something lacking there. It seems to me like there might be a way to address that through landscape architecture.

Q: What’s something you learned while at ASU — in the classroom or otherwise — that surprised you, that changed your perspective?

A: My internship (with ADOT), being part of the meetings with the different communities. They involved me in some of the pre-design stuff, going to meetings with different communities the projects run through. One thing they do is try to collaborate with the different communities to take into consideration their goals, doing community workshops and those kinds of things. …. You have to take other people’s culture into consideration, not just do it because that’s how you think it should be. The back and forth that goes on surprised me; it’s always a challenge. You have a meeting and then you go and design something and you send it back and there’s some other thing that wasn’t thought of before and then you have to meet again. It’s not simple.

Q: Why did you choose ASU?

A: I wanted to stay close to my family, my sister and my daughter, AnnaMariah, who’s 10.

Q: What’s the best piece of advice you’d give to those still in school?

A: Just to look to surround yourself with people who will support your ideas and can also be critical but not put you down.

Q: What was your favorite spot on campus, whether for studying, meeting friends or just thinking about life?

A: I didn’t do a lot of hanging out on campus. There are several courtyards around campus that I’ve seen and really enjoy a lot (Mary Lou Fulton). The one on the south side of the MU, with the fountain, I like that.

Q: What are your plans after graduation?

A: I want to take my daughter to Mexico. She hasn’t met my family down there. I’m planning to go there for 10 days. My daughter really likes the beach, so the plan is to hang out with my family for five days and then go to different beaches for five days. Besides the fun, I need to be working. I hope to be working. I’m applying for jobs once I’m done graduating. I can work at ADOT for six months after I graduate so I have a bit of a cushion. I will be sending my resume out right after graduation.

Q: If someone gave you $40 million to solve one problem on our planet, what would you tackle?

A: I just started learning about this thing called microaggression so maybe that. It’s just a stigma that people put on different groups of people that is negative.

Q: Anything you want to add?

A: My mom: I want to find a way to thank her. I see the love of God in her, I think that she’s never given up on me.

When I needed her she was there, and when I thought I didn’t need her, she was still there. Moms are special. Especially now — when I came to ASU I knew what was going to happen, that things were going to get difficult and I was going to need help with my daughter. I talked with my stepdad and my mom and told them I might need help. After the second year, she moved down here to help me out, and they got a second home. She’s been very supportive. I probably wouldn’t have been able to finish without her.

Q: What’s the most important lesson your mom taught you?

A: [He thinks for a long moment.] Love. Just love.

Written by Deborah Sussman Susser and Brandon Chiz; top photo by Charlie Leight/ASU Now