lunes, 15 de octubre de 2007

New Force-fluorescence Device Measures Nanometer-scale Motion

Science Daily — A hybrid device combining force and fluorescence developed by researchers at the University of Illinois has made possible the accurate detection of nanometer-scale motion of biomolecules caused by pico-newton forces.

"By combining single-molecule fluorescence resonance energy transfer and an optical trap, we now have a technique that can detect subtle conformational changes of a biomolecule at an extremely low applied force," said U. of I. physics professor Taekjip Ha, the corresponding author of a paper to appear in the Oct. 12 issue of the journal Science.

The hybrid technique, demonstrated in the Science paper on the dynamics of Holliday junctions, is also applicable to other nucleic acid systems and their interaction with proteins and enzymes.
The Holliday junction is a four-stranded DNA structure that forms during homologous recombination -- for example, when damaged DNA is repaired. The junction is named after geneticist Robin Holliday, who proposed the model of DNA-strand exchange in 1964.
To better understand the mechanisms and functions of proteins that interact with the Holliday junction, researchers must first understand the structural and dynamic properties of the junction itself.
But purely mechanical measurement techniques can not detect the tiny changes that occur in biomolecules in the regime of weak forces. Ha and colleagues have solved this problem by combining the exquisite force control of an optical trap and the precise measurement capabilities of single-molecule fluorescence resonance energy transfer.
To use single-molecule fluorescence resonance energy transfer, researchers first attach two dye molecules -- one green and one red -- to the molecule they want to study. Next, they excite the green dye with a laser. Some of the energy moves from the green dye to the red dye, depending upon the distance between them. The changing ratio of the two intensities indicates the relative movement of the two dyes. 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 university's 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; graduate students Ruobo Zhou, Michelle Nahas and Jin Yu; and molecular biology professor David M. J. Lilley at the University of Dundee, UK.
The work was funded by the National Science Foundation and the National Institutes of Health.


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