O
ver the past several decades, biologists and chemists have amassed reams of data about the natural world by investigating its properties at the molecular level. However, a majority of such studies have focused on collections of molecules that typically number in the billions. Because molecules can change their behavior on the basis of interactions with their nearby neighbors, such studies can be misleading—they sum up a population’s average properties but give little insight on how molecules behave as individuals. Biophysicist Taekjip Ha of the University of Illinois at Urbana–Champaign is working to fill this knowledge gap by employing a variety of techniques, including pioneering use of single-molecule FRET. By characterizing single biomolecules, he is magnifying scientific understanding of some pivotal molecular components of life. From Physics to Biochemistry Ha was born in 1968 in Seoul, South Korea. With both parents as teachers, he quickly learned the importance of a good education. He strived to excel as a student, regardless of the subject matter he studied. Though not particularly focused on science as a child, Ha decided in high school to concentrate his efforts on gaining admission to the physics department of Seoul National University (SNU). “People who got good scores on South Korea’s national college entrance exam said that’s where they were going,” recalls Ha. “I said, wow, that’s what I have to do. I have to go to the physics department to be with these really smart people.” Against his parents’ initial wishes— they’d encouraged him early on to become a doctor or lawyer—Ha began his physics www.acschemicalbiolog y.o rg
studies at SNU in 1986. He filled his curriculum with physics and mathematics courses. He remembers a class called mathematical analysis as his favorite. The course involved using fundamental theorems to “construct your own mathematical universe,” says Ha, a line of study that taught him logical thinking. He continues to use this skill to set up and analyze his current experiments. Early in his undergraduate studies, Ha set a goal for himself to attend a university in the United States to pursue his doctoral degree. By the time he graduated in 1990, he had gained admission to a Ph.D. program in physics at the University of California, Berkeley. He chose the school for its highly ranked physics program and its large department. “I thought that perhaps I could have more choices with research,” he says. Ha started his studies with a plan to do theoretical research in condensed-matter physics, a discipline that studies the materials that make up semiconductors and superconductors. However, after attending a course by experimentalist Eugene Cummins, who studies atomic physics, Ha switched his own focus to experimental study. Soon afterward, he joined the lab of Raymond Jeanloz in Berkeley’s geophysics department. There, he worked on a project to place nitrogen and carbon under very high pressures, with the goal to create a material harder than diamonds. After several months, Ha and his colleagues had little success with the project. Taking a temporary leave of absence from Berkeley, he returned to South Korea for a year to fulfill South Korea’s military service requirements. Upon his return, Ha joined the lab of Daniel Chemla, a prominent
Image courtesy of Taekjip Ha
Ready for Their Close-Ups: Investigating Single Molecules
Profiles provide insights into the lives, backgrounds, career paths, and futures of scientists who serve as Experts on ACS Chemical Biology’s online Ask the Expert feature. Dr. Ha will begin answering your questions in mid-January, 2007. Readers are encouraged to submit questions to the Experts at www.acschemicalbiology.org. The editors will post the most interesting exchanges on the web site.
Published online December 15, 2006 10.1021/cb6004799 CCC: $33.50 © 2006 by American Chemical Society
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“There were so many things I wanted to study that I couldn’t do it on my own. I figured it was time to start my own group.”
physicist known for his studies of quantum optics of semiconductors. Soon after joining Chemla’s group, Ha began working closely with then-staff scientist Shimon Weiss. Recognizing that Ha had a talent for assembling machines, ranging from switchboxes to more complicated instruments, Weiss suggested that he take advantage of this skill to build a near-field scanning optical microscope, a machine equipped with a small aperture and a short-pulse laser able to measure a material’s properties with high time and spatial resolution. Once Ha finished building the machine, he originally planned to use it to study semiconductors. His initial experiments were not successful. However, Ha notes that his seeming failure was actually a turning point for his career. Researching other uses for his new device, he found a few papers that discussed utilizing the machine to measure the properties of single fluorescent molecules. Ha immediately grasped the significance of such research. “Most of the knowledge we have about chemistry, biology, and physics, we got by studying molecules in the billions and measuring their average properties,” he says. “But looking at single molecules, you have the ability to gain a much deeper understanding of what’s going on.” Because other groups were already collecting measurements of single fluorescent molecules, Ha decided to add a more complicated twist to his own work by collecting information with FRET. In this technique, an excited donor dye molecule transfers energy to an acceptor molecule, providing information about their interaction. To bring the two molecules into proximity, Ha, Weiss, and their colleagues worked with Berkeley scientist Paul Selvin, who had successfully attached dye molecules to segments of single-stranded DNA (ssDNA). When complementary strands hybridized, the two dye molecules were in proximity to each other. 742
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In a proof-of-concept experiment, Ha and his colleagues used the technique to measure FRET between a single pair of donor and acceptor molecules, tetramethylrhodamine and Texas red. The researchers published their work in 1996 (1). Four Focuses Ha published several related papers before completing his doctoral degree in 1996. “I ended up doing pretty well in terms of publications,” he recalls. “You could publish fairly simple experiments because everything in this area was so new.” Ha remained in Weiss and Chemla’s lab an additional year doing postdoctoral work and training other graduate students in the methods he used. In 1997, he accepted a second postdoctoral fellowship in the lab of Steven Chu at Stanford University. Days after Ha joined the new group, Chu won the Nobel Prize in Physics. Chu’s award and additional responsibilities left Ha and other lab members with unexpected free reign over their projects. Though Ha had initially been charged with using an atomic force microscope to measure conformational changes in enzymes, he decided to add his own twist based on his previous research: he combined atomic force microscopy with FRET, using quantum dots applied to the microscope’s tip as donor molecules and attaching acceptor molecules to his samples. The technique had only limited success, but the fluorescent part of the instrument was useful for observing the folding of single RNA molecules, which he studied in collaboration with James Williamson at the Scripps Research Institute. Ha published his first paper in Chu’s lab on RNA folding in 1999 (2). After this publication, Ha searched for a new system on which to build his own independent research. He found an ideal candidate in helicase, an enzyme that unwinds double-stranded DNA (dsDNA). After reading a special review issue of Cell on motor proteins, Ha realized that helicase Brownlee
was then the only remaining motor protein mentioned in the reviews that had not been studied at the single-molecule level. Ha began attempting to measure helicase’s unwinding activity using FRET. However, the enzyme stuck to the glass surface of his lab dishes, hindering its study. Collaborators in Stanford’s chemistry department, including Chris Chidsey, suggested using a polymer brush to prevent adhesion. The problem was solved, giving Ha the impetus to move his work to an independent lab. “There were so many things I wanted to study that basically I couldn’t do it on my own. I figured it was time to start my own group and hire people to work on all these different ideas,” he says. In 2000, Ha accepted an offer for an assistant professorship at the University of Illinois at Urbana–Champaign, where he developed four broad areas on which to focus his research: the enzyme helicase, a DNA recombination intermediate called the Holliday junction, a recombinationmediating protein called RecA, and a class of proteins called SNARE, an acronym for soluble NSF attachment receptor, that mediate membrane fusion. Single Molecules in Action Ha realized that researchers knew little about how helicase unwinds dsDNA. He and his colleagues, including collaborator Tim Lohman of Washington University, were curious about whether the molecule worked as a monomer to unwind DNA or whether only a dimer could complete the task. Using FRET, the researchers investigated this problem. They published compelling evidence in 2002 suggesting that helicase was only active as a dimer. The team’s research showed that if one monomer of the pair dissociates from DNA, unwinding stalls (3). Recently, Ha’s team published another paper outlining a quirk in helicase’s mechanism (4). Using FRET again, the researchers w w w. a c s c h e m i ca l biology.org
found that if helicase is obstructed in its path along duplex DNA, then the molecule will quickly snap back to its original location to repeat its sweep over the same stretch of DNA. Ha says that his team is not sure of the biological significance of this activity. However, their hypothesis is that helicase could be performing an essential housekeeping function for DNA. “Helicase moving on a single strand of DNA many times could be used to prevent the accumulation of unwanted proteins on a DNA segment. Like a snowplow, it continues to sweep over and over again,” explains Ha. About a third of his lab members continue to study helicase. Another system that Ha and his colleagues are investigating is the Holliday junction, a four-stranded DNA molecule that forms as an intermediate during recombination. Researchers had known that this structure can have two alternatively folded conformations and that it could convert between these two states. However, notes Ha, scientists had not been able to observe this conversion in real time. In collaboration with David Lilley of the University of Dundee in Scotland, Ha and his colleagues followed the molecule’s changes between isomers, research they published in 2003 (5). More recently, the team has used optical tweezers to further investigate the junction’s conformational changes under gentle tension. A third project on which Ha and his team are focused involves RecA, a protein that catalyzes pairing of ssDNA with complementary regions of dsDNA during recombination. This protein filament is highly dynamic in cells, growing and shrinking by adding or subtracting RecA monomers as it accomplishes its task. Using FRET, Ha’s group investigated this process. The researchers found that about five monomers are necessary to start the growth of a RecA filament. Additionally, their work suggested that this filament grows and shrinks one monomer at a time (6 ). www.acschemicalbiolog y.o rg
“Our measurements for the first time gave concrete numbers for this highly dynamic process,” says Ha. He and his team recently began studying the SNARE family of proteins, a group thought to play pivotal roles in the membrane fusion necessary for exocytosis and vesicle trafficking in the neural junction and elsewhere. The researchers were interested in looking at the proteins’ action at the single-vesicle level. Ha’s team developed an assay in which they created vesicles with donor or acceptor fluorescent dyes incorporated into their membranes. When these donor and acceptor vesicles mix, they emit a FRET signal. Using this assay, Ha’s team, in collaboration with Yeon-Kyun Shin of Iowa State University, has found that vesicles do not fuse in a single step. Rather, the SNARE proteins guide vesicle membranes through several intermediates (7 ). Their initial findings used yeast SNARE proteins as a model. However, Ha notes that his team’s current work focuses on the proteins’ action in neurons. The Future of FRET Though he and his colleagues have made important contributions to the four systems he initially set out to study, Ha notes that his work is far from complete. He is cultivating several ideas for future experiments using FRET and other techniques. “So far, we’ve been doing relatively simple experiments for single-molecule measurements, such as the interaction between one protein and one DNA. In terms of the future, we’ll have to make the system more relevant biologically, because in the cell, proteins don’t function in isolation,” he says. “There are interactions between multiple components.” He adds that his group is planning to make FRET even more useful by developing techniques that involve multiple colors of fluorescent dyes. He also plans on continuing to push the boundaries
of his research through incorporating optical tweezers and other technologies. Additionally, he would like to study other biological systems, such as ribosomes and chromatin remodeling. Ha notes that this combination of unique techniques and a novel focus will probably continue to turn up interesting aspects of biological phenomena. “I’m interested in studying things that will make a difference—problems or approaches that are not being pursued elsewhere,” he says. “This way, hopefully whatever we discover will be quite unique in terms of contribution to the field.” —Christen Brownlee, Science Writer
REFERENCES
1. Ha, T., Enderle, T., Ogletree, D. F., Chemla, D. S., Selvin, P. R., and Weiss, S. (1996) Probing the interaction between two single molecules—fluorescence resonance energy transfer between a single donor and a single acceptor, Proc. Natl. Acad. Sci. U.S.A. 93, 6264-6268. 2. Ha, T., Zhuang, X., Kim, H. D., Orr, J. W., Williamson, J. R., and Chu, S. (1999) Ligand-induced conformational changes observed in single RNA molecules, Proc. Natl. Acad. Sci. U.S.A. 96, 9077-9082. 3. Ha, T., Rasnik, I., Cheng, W., Babcock, H. P., Gauss, G. H., Lohman, T. M., and Chu, S. (2002) Initiation and reinitiation of DNA unwinding by the Escherichia coli Rep helicase, Nature 419, 638-641. 4. Myong, S., Rasnik, I., Joo, C., Lohman, T. M., and Ha, T. (2005) Repetitive shuttling of a motor protein on DNA, Nature 437, 1321-1325. 5. McKinney, S. A., Declais, A. C., Lilley, D. M. J., and Ha, T. (2003) Structural dynamics of individual Holliday junctions, Nat. Struct. Biol. 10, 93-97. 6. Joo, C., McKinney, S. A., Nakamura, M., Rasnik, I., Myong, S., and Ha, T. (2006) Real-time observation of RecA filament dynamics with single monomer resolution, Cell 126, 515-527. 7. Yoon, T. Y., Okumus, B., Zhang, F., Shin, Y. K., and Ha, T. (2006) Multiple intermediates in SNAREinduced membrane fusion, Proc. Natl. Acad. Sci. U.S.A., in press.
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