A new route to biological engineering might now be possible thanks to work by researchers at the University of Illinois at Urbana-Champaign who have observed the structure and the correlated function of specific proteins involved in DNA repair.
The DNA repair enzyme helicase UvrD from Escherichia coli has an "open" or "closed" conformation the activities of which have been revealed by the team, using optical traps and single-molecule fluorescence microscopy, to look at form and dynamic function simultaneously. Taekjip Ha worked with optical traps expert Yann Chemla and their colleagues on combining the two techniques to give them definitive answers relating protein form to function. [Science, 2015, 348(6232), 352-354; DOI: 10.1126/science.aaa0130 and 344-347; DOI: 10.1126/science.aaa0445]
Helicase UvrD works by separating damaged DNA strands but scientists debated whether one or two proteins were involved in this process. "To answer that question, we put a fluorescent dye molecule on each protein so we could count them," explains Chemla. "Then we watched the unwinding with an optical trap." The team showed a single UvrD can unwind the DNA but only so far, oscillating back and forth in "frustrated" activity. With two UvrD molecules, the process goes much further and doesn't oscillate.
Then, using smFRET (single-molecule fluorescence resonance energy transfer), in which the team adds two dyes to the molecule, they were able to measure separation and determine whether the protein is open or closed. "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," Chemla adds.
An additional experiment carried out in the Ha laboratory involved engineering a structural homolog, Rep, which can be locked into the closed position when it then becomes a "superhelicase" capable of unwinding double-stranded DNA over a much great reach. Locked in the open position, this helicase is inactive. The team repeated this experiment on yet another homolog, PcrA, with a similar positive result but with the added benefit of being able to "lock" this protein in the closed state using another protein.
"Proteins are flexible," Chemla told Materials Today. "each may serve multiple functions. The presence of other proteins can determine which function is active by changing its structure." Such engineering of enzymes holds the promise of creating molecules for specific tasks that might be used in rapid DNA sequencing using nanopore technology, for instance.
David Bradley blogs at Sciencebase Science Blog and tweets @sciencebase, he is author of the popular science book "Deceived Wisdom".