To monitor the transition, Weninger and student John Sakon first analyzed the crystal structure of the SNARE complex in order to create two dual-labeled forms of SNAP-25, one with two fluors (Cy3, the donor, and Cy5, the acceptor) separated by 1.5 nm (a “high-FRET” variant) in the folded conformation, and one where the distance was 8 nm (“low-FRET”).

“What we often do is we take the crystal structure … and then we design places to put FRET labels based on that structure,” explains Weninger. “Then we watch a real protein move and we can interpret the FRET signal as changes away from that conformation.”

To watch that movement, Weninger and Sakon injected relatively small numbers of the proteins into mammalian cells and tracked individual molecules using TIRF microscopy in a process known as single-molecule FRET (smFRET). This single-molecule (as opposed to “ensemble”) approach allows studies on the behavior of individual molecules, each of which may have distinct properties, rather than the averaged behavior of all labeled molecules. “The benefit of single molecules is that it gives you sensitivity to [molecular] subpopulations,” notes Weninger. Another benefit of smFRET: the ability to witness “transient dynamic processes” — that is, unsynchronized molecular outliers.

The flipside, Weninger says, is that smFRET requires a brighter excitation source and more sensitive detectors. Those effects combine to raise experimental background, making signal — already just a fraction of that in an ensemble experiment — harder to detect. At the same time, those signals are fleeting, as the bright excitation induces more rapid photobleaching. “The question is whether you can measure those smaller signals in the context of the experiment,” he says.

In this case, Weninger and Sakon were able to measure both the speed of folding — they detected fluorescence emission from the FRET acceptor less than a second after injection — and the trajectories (that is, motion) of individual molecules. Their results showed that SNAREs from distinct cellular trafficking pathways readily substitute for each other in vivo. “The single-molecule approach provides insights into these transient pathway-crossing events,” Weninger says.

Ensemble Fret

While Weninger and Sakon are focused on understanding the folding properties of single protein molecules, Gruebele's group uses FRET to study all the labeled proteins in a cell en masse, an ensemble measurement that works because he synchronizes their folding behavior with heat.

Gruebele's heat source is an infrared laser tuned to excite the molecular bonds in water. By applying the laser to rapidly heat and cool cells expressing temperature-sensitive proteins tagged on either end with thermostable fluorescent proteins (in this case, a GFP donor and mCherry recipient), he can correlate changes in FRET efficiency between the two fluors with the degree to which the protein between them is folded in vivo.

“The [temperature] jump gets the protein to fold or unfold, while most other proteins are unaffected,” says Gruebele. He likens the process to giving cells “a slight fever.”

Gruebele and his team applied their approach, called “fast relaxation imaging” (FreI), to a “low-melting-temperature triple mutant” form of the metabolic enzyme, phosphoglycerate kinase (PGK) in human cells. Cells expressing labeled PGK variants were imaged on glass slides using a customized inverted microscope with lasers for heating as well as exciting both donor and acceptor fluors. Folding was assessed colorimetrically, by assessing the relative intensity of red and green fluorescence in transfected cells. Increasing the temperature in this proof-of-principle experiment from 27oC to 31oC had no effect on FRET efficiency (that is, the cellular color remained static), as the protein remains stable in this temperature range. But bumping the thermostat from 39oC to 43oC resulted in protein unfolding within seconds — an effect the team could resolve pixel by pixel.

With proof-of-principle in hand, the team is now looking to assess how protein stability and dynamics vary throughout the cell. According to Gruebele, preliminary results indicate that proteins fold faster and are more stable in the nucleus than in the cytoplasm — the protein's “melting temperature” in vitro is about 38oC, compared to 43oC in the nucleus and 41oC in the endoplasmic reticulum. “That seems like a relatively small temperature difference,” Gruebele notes. “But a human being at 38oC is in good shape; at 43oC you are on your death bed.”