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New force-fluorescence device measures motion previously undetectable
Date:10/11/2007

. Therefore, by monitoring the brightness of the two dyes, the researchers can determine the motion of the molecule.

The optical trap, on the other hand, functions somewhat like the fictional tractor beam in Star Trek. In this case, a focused laser beam locks onto a microsphere attached to one end of the molecule to be studied. The optical trap can then pull on the molecule like a pair of tweezers.

By combining the two techniques, we get the best of both worlds, said Ha, who also is an affiliate of the universitys Institute for Genomic Biology and of the Howard Hughes Medical Institute. Using the optical trap, we can pull on DNA strands with forces as small as half a pico-newton. Using single-molecule fluorescence resonance energy transfer, we can measure the resulting conformational changes with nanometer precision.

By probing the dynamics of the Holliday junction in response to pulling forces in three different directions, the researchers mapped the location of the transition states and deduced the structure of the transient species present during the conformational changes.

Based on our previous studies, we knew the Holliday junction fluctuated between two structures, Ha said, but how it moved from one place to the other, and what intermediates were visited along the pathway, were unknown.

With this latest work, the researchers have deduced the pathway of the conformational flipping of the Holliday junction, and determined the intermediate structure is similar to that of a Holliday junction bound to its own processing enzyme.

The next challenge is to obtain a timeline of movement by force, for example, due to the action of DNA processing enzymes, and correlate it with the enzyme conformational changes simultaneously measured by fluorescence, Ha said.

With Ha, co-authors of the paper are former U. of I. postdoctoral research associate and lead-author Sungchul Hohng (now at Seoul National University); physics professor Klaus Schulten; gr
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Contact: James E. Kloeppel, Physical Sciences Editor
kloeppel@uiuc.edu
217-244-1073
University of Illinois at Urbana-Champaign
Source:Eurekalert

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