Kapanidis, though, has devised an approach he thinks is relatively scalable: switchable FRET. “Switchable” fluorophores are dyes that can be turned on and off using different wavelengths of light and/or chemicals. By placing one FRET donor and two acceptors on a single biomolecule and then cycling between the different acceptors (that is, switching them on and off in sequence), Kapanidis and his team showed that they could probe the structure and dynamics of both a protein-DNA complex and a Holliday junction.
Now Kapanidis' team is working to improve the method. “Given our current FRET resolution, we anticipate that extensions to more than two pairs will be straightforward,” he wrote (2). For instance, he continued, the technique could facilitate analysis of multimeric protein complexes, such as helicases.
Going In VivoKeith Weninger, associate professor of physics at North Carolina State University, is addressing another problem with smFRET: moving the technique from the in vitro to the in vivo world.
“In vitro biochemistry has been very powerful and has added to our knowledge, but there's a lot of evidence that the environment inside cells is important in controlling the function of many proteins,” says Weninger.
Indeed, Weninger's interest may be summed up elegantly as “the dynamics of proteins in vivo,” but that's easier said than done. smFRET in vivo is a tricky business. Among the problems are high background fluorescence and a difficulty labeling molecules in vivo—this is because fluorescent proteins make for poor smFRET partners photophysically, and also because it's difficult to control their concentration.
Weninger, though, has found a way to circumvent those issues. Earlier this year, he and graduate student John Sakon showed it was possible to use intramolecular FRET to capture the conformational dynamics of SNARE proteins during membrane fusion inside living cells. His solution: microinjecting prelabeled, recombinant protein.
“We typically would inject about 15 cells in 30 minutes per plate,” he says. This enabled the team to track dozens of molecules per plate before the fluorophores fizzled out. To overcome the fluorescent background, Weninger and his team optimized both growth conditions and their microscopy protocols. For instance, they used fluorescent dyes that were particularly bright. And instead of standard TIRF, his team used “near-TIRF,” in which the sample is illuminated at very steep angles to probe different optical planes without overwhelming the system with light.
This is certainly not the first time FRET has been applied in vivo. The technique has been used for years to identify protein-protein interactions in living cells, but those experiments measure bulk populations, not individual molecules. In 2004, a Japanese team used smFRET to measure protein–small molecule interactions in vivo (3), but that was a colocalization experiment. “This is the first demonstration of single-molecule in vivo detection of conformational changes,” Weninger says.
Given the pace of developments in this field, it surely won't be the last. And other developments are on the horizon, too. Ha, for instance, is developing a method he describes as the “third way” between in vitro and in vivo studies, to study the biochemistry of native cellular (as opposed to recombinant) complexes. “It's working,” he says, “and I think you'll see many more studies of this type in the future.” And though Life Technologies is keeping mum about a formal release date for Starlight, Beechem sees promise. “I think it's the most advanced FRET system that's ever been made.”
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