New laboratory technique simultaneously reveals structure and function of proteins critical in DNA repair

Siv Schwink
4/17/2015

Professor Taekjip Ha
Professor Taekjip Ha
Professor Yann Chemla
Professor Yann Chemla

 

 

 

 

 

 

 

 

 

 

 

By combining two highly innovative experimental techniques, scientists at the University of Illinois at Urbana-Champaign have for the first time simultaneously observed the structure and the correlated function of specific proteins critical in the repair of DNA, providing definitive answers to some highly debated questions, and opening up new avenues of inquiry and exciting new possibilities for biological engineering.

Scientists who study biological systems at the molecular level have over the years looked to the structure of protein molecules—how the atoms are organized—to shed light on the diverse functions each performs in the cell. The inverse is also true: observing the specific work particular protein molecules perform has provided important clues as to the conformation of the respective molecules. But until recently, our most advanced laboratory experiments could only investigate one at a time—static form or dynamic function—and from the results, deduce the other. This indirect method often doesn’t provide definitive answers.

Now Illinois biological physicists Taekjip Ha and Yann Chemla have combined two cutting-edge laboratory techniques that together directly get at the structure-function relationship in proteins. Ha is well recognized for his innovative single molecule fluorescence microscopy and spectroscopy techniques. Professor Yann Chemla is a top expert in optical trapping techniques. Their combined method—simultaneous fluorescence microscopy and optical trapping—yields far more definitive answers to questions relating structure to function than either technique could independently.

Working in collaboration, Ha and Chemla each applied the above techniques in their laboratories, with conclusive results. The findings of these experiments have been published in two separate articles in the April 17 issue of the journal Science.

Chemla’s lab team looked at the structure-function relationship in the helicase UvrD, a protein, found in the bacterium E. coli, that separates strands of DNA in need of repair by unwinding and unzipping them. There is an equivalent protein that performs the same vital process in humans. The first question Chemla’s team investigated was how many UvrD proteins are required to perform this task—recent debates among scientists have the number at either one or two.

“The way we answered this,” explains Chemla, “we put a dye molecule on each protein with fluorescence—so we could count them. Then we watched the unwinding with an optical trap. We found that a single UvrD helicase can do something—it unwinds the DNA, but not very far. It just goes back and forth a small distance, so we call it ‘frustrated’. When we have two UvrD molecules, it seems to unwind much further and doesn’t go back and forth as much.”

The DNA repair helicase UvrD can exist in an “open” (green, blue, cyan, and gray colored protein, upper right) or “closed” (middle) conformation. An instrument combining optical traps (red cones) and a single-molecule fluorescence microscope (green) is used to measure directly the relationship between these two structural states and their respective functions on DNA. Image by Matt Comstock, University of Illinois at Urbana-Champaign
The DNA repair helicase UvrD can exist in an “open” (green, blue, cyan, and gray colored protein, upper right) or “closed” (middle) conformation. An instrument combining optical traps (red cones) and a single-molecule fluorescence microscope (green) is used to measure directly the relationship between these two structural states and their respective functions on DNA. Image by Matt Comstock, University of Illinois at Urbana-Champaign
Chemla’s team also resolved one question on the structure-function relationship in UvrD. There are two distinct structures or states that are associated with UvrD, with the molecule organized in either an “open” or “closed” position. The function associated with each state has been debated among experts for several years.

“This time, we used smFRET (single-molecule fluorescence resonance energy transfer). We put two dyes on the molecule, and based on the distance between them, we could see one or another color of light, indicating whether the molecule was in the open or closed position. Then we used an optical trap to observe whether the molecule was unwinding the double-stranded DNA.

“We found that the molecules actually swiveled from open to closed and back again. As it turns out, the closed state unwinds the strands, using a torque wrench action. The open state allows the strands to zip together.”

This work is published in the article “Direct observation of structure-function relationship in a nucleic acid­–processing enzyme” in Science (April 17, 2015, v. 348, no. 6232, pp. 352-354; DOI: 10.1126/science.aaa0130).

In Ha’s laboratory, the team engineered a structurally homologous helicase protein called Rep, fastening it in either the closed or the open state by using a cross-linking molecule as “tape”.

The team found that when locked in the closed position, it becomes a “superhelicase” capable of unwinding double-stranded DNA over a great distance. Locked in the open position, the helicase was defunct—it performed no task.

The ability to bioengineer molecules to perform specific tasks holds promise for applications, including rapid DNA sequencing with nanopore technology.

Ha comments, “The superhelicase we engineered based on our basic, fundamental understanding of helicase function can be used as a powerful biotechnological tool for sensitive detection of pathogenic DNA in remote areas, for example.”

The team repeated the experiment on another structurally homologous helicase protein called PcrA, with the same result. Here, the team was further able to show how another protein that interacts with PcrA and is known to contribute to its effectiveness physically achieves that purpose. The team was able to demonstrate that the addition of the protein locked PcrA in the closed state.

“Proteins are flexible,” explains Chemla. “Each may serve multiple functions. The presence of other proteins can determine which function is active by changing its structure.”

This work is published in the article “Engineering of a superhelicase through conformational control” in Science (April 17, 2015, v. 348, no. 6232, pp. 344-347; DOI: 10.1126/science.aaa0445).

This research was funded by the National Science Foundation through Physics Frontier Center grants (PHY 0822613 and PHY 1430124) and through a CAREER grant (MCB 09-52442 to YRC), and by the National Institutes of Health grants (R01 GM065367 to TH; R21 RR025341; and R01 GM045948 to TML), and by a fellowship through the Alfred P. Sloan Research Foundation (to YRC).

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Associate Head for Graduate Programs and Professor S. Lance Cooper has been awarded the 2018 Excellence in Graduate Student Mentoring Award of the Office of the Provost at the University of Illinois at Urbana-Champaign.

One of the Campus Awards for Excellence in Instruction conferred annually at the campus’s Celebration of Teaching Excellence, this accolade recognizes sustained excellence in graduate student mentoring; innovative approaches to graduate advising; major impact on graduate student scholarship and professional development; and other contributions in the form of courses and curricula, workshops, or similar initiatives. Cooper was presented with the award on April 12, 2018.

The University of Illinois has received a three-year, $1 million grant from the Alfred P. Sloan Foundation to continue funding for the Sloan University Center of Exemplary Mentoring at Illinois. The program, started in 2015, supports underrepresented minority doctoral students in science, technology, engineering and math fields and is one of nine UCEMs throughout the country.

The UCEM emphasizes mentoring, professional development and social activities to build a community of scholars. The center hosts an extensive orientation program for new students, workshops and seminars in addition to financial support in the form of scholarships. The center also works with departments to set up a mentoring team for each scholar and monitors academic and research progress.

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Sir Anthony Leggett, winner of the 2003 Nobel Prize in Physics and the John D. and Catherine T. MacArthur Professor of Physics at the University of Illinois at Urbana Champaign, turned 80 years old on March 26. To celebrate, the Department of Physics is hosting a physics symposium in his honor, with participants coming from around the world. The symposium, “AJL@80: Challenges in Quantum Foundations, Condensed Matter Physics and Beyond,” is targeted for physicists and requires pre-registeration. It begins tonight, Thursday evening, and will go through Saturday evening (March 29 – 31, 2018).

In conjunction with the symposium, two public presentations will be offered back-to-back on Friday, March 30, starting at 7:30 p.m., at the I Hotel and Conference Center’s Illini Ballroom. (1900 S. First St., Champaign). There is no admission fee and registration is not required—all are welcome.

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