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SBKB [doi:10.1038/sbkb.2011.13]
Technical Highlight - April 2011
Short description: A trio of new approaches pushes forward the utility of single-molecule imaging.

Single-molecule FRET gets a boost from three new approaches. Netweb01 at commons.wikimedia.org/.

Single-molecule fluorescence resonance energy transfer (smFRET) has been invaluable to the study of the behavior of molecules, allowing researchers to observe the structural conformations taken by individual molecules in response to different conditions. However, the full promise of smFRET has yet to be realized owing to technical challenges associated with the nature of the approach. Three recent studies address these difficulties, increasing the applicability of smFRET.

One obstacle that hinders the progress of smFRET is its low time resolution. In theory, smFRET should allow for a 10-μs resolution during imaging, but practical applications usually see resolution in the range of milliseconds. This limit makes it difficult to observe and quantify submillisecond processes such as protein folding. Muñoz and colleagues addressed one of the major causes of this loss of resolution: excitation of the dye into a high-energy triplet state, which depletes the reservoir of ground-state molecules and bleaches the dye. This process, called blinking, results in substantially reduced temporal resolution. To combat this, the authors used dissolved oxygen, which efficiently quenches triplets but can also form highly reactive species. The authors tested several compounds and, ultimately, cysteamine was found to scavenge the reactive oxygen derivatives efficiently with minimal fluorescence quenching, and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was also added to further quench unwanted triplets. The end result was 50-μs to 100-μs resolution of the folding of the BBL protein and α-spectrin SH3 domain.

In the second study, Deniz and colleagues used a microfluidics approach to also improve resolution. Combining microfluidics with smFRET imaging has allowed researchers to examine molecules in response to changing conditions, but current microfluidics devices that are optimized for smFRET do not allow for rapid imaging in this context. The authors solved this problem with a specially designed microfluidic device that mixes the single molecules being studied with two high-speed buffer streams. The device then rapidly slows the molecules, allowing for imaging within approximately 200 μs of mixing, eliminating much of the dead time and giving insights into conformational changes and folding biophysics that were until now difficult to observe. By examining the behavior of the intrinsically disordered protein α-synuclein exposed to a lipid mimic in their system, the authors followed the rapid time evolution of an encounter complex. They saw that a 'broken helix' conformation that the protein can take may actually be a relevant part of the folding process and not an artifact (as has been previously suggested). In addition, the authors note that the unfolded-to-folded and folded-to-unfolded transitions occured via different observed pathways, under non-equilibrium folding and unfolding conditions, respectively.

A second major challenge for smFRET is the difficulty in carrying out high-throughput analysis. Now, Majumdar and colleagues have adapted the microfluidics approach for rapid, high-throughput assessment of single-molecule behavior. The authors utilized a microfluidic mixing ring that uses one pump for the injection of reagents and a second pump for mixing. Previous ensemble approaches did not allow for distinction of specific subpopulations in the sample, something that is now possible with this approach. The authors demonstrated this with single-stranded DNA exposed to varying salt and polyadenine concentrations, and with mRNA synthesized by RNA polymerase in response to changing glutamate conditions.

These three new approaches have opened the door for the examination of molecules using smFRET at levels that were previously inaccessible, in terms of both time and scale. Further development—perhaps even combination of these approaches—will increase the utility of smFRET and our understanding at the molecular level.

Steve Mason

References

  1. L.A. Campos et al. A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy.
    Nat. Methods 8, 143-146 (2011). doi:10.1038/nmeth.1553

  2. Y. Gambin et al. Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing.
    Nat. Methods 8, 239-241 (2011). doi:10.1038/nmeth.1568

  3. S. Kim et al. High-throughput single-molecule optofluidic analysis.
    Nat. Methods 8, 242-245 (2011). doi:10.1038/nmeth.1569

  4. M. Gruebele A triple threat to single molecules.
    Nat. Methods 8, 213-215 (2011). doi:10.1038/nmeth0311-213

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