The role of SNARE proteins in a cellular process known as exocytosis. Image from http://en.wikipedia.org/wiki/File:Exocytosis-machinery.jpg
Proteins are great believers in hands-on teamwork. They come together in groups to directly interact with each other and set off reactions. But because these interactions happen over extremely tiny distances, watching them has been almost impossible. In a paper recently out in the Journal of the American Chemical Society, researchers described a method that allows them to track multiple proteins that closely interact with each other below the optical diffraction limit at the single-molecule level.
The optical diffraction limit has been the long-standing problem of optical microscopy because it stops researchers from seeing events that happen at distances less than 200 nm, the limit of light diffraction. In the past decade, optical techniques have emerged that allow researchers to bypass this limit. The new techniques, such as single-molecule fluorescence resonance energy transfer (FRET), have allowed researchers to view molecules on the scale of a few nanometers, distances that often take place in biological systems.
“But the single-molecule FRET technique has been able to follow only one protein complex,” explains Tae-Young Yoon at KAIST in South Korea, and multiple protein complexes within one diffraction-limited spot cannot be distinguished from one another.
So Yoon and colleagues developed a way to use single-molecule FRET on multiple proteins below the diffraction limit. As their proof of concept, they used SNARE proteins, which are involved in the fusion of lipid membranes. “It is now believed that, with only few exceptions, membrane fusion processes in eukaryotic cells are all mediated by the SNARE proteins, which underscores the importance of the SNARE family and their complex formation,” says Yoon. “Moreover, it has been long anticipated that SNARE proteins are in a clustered state and that multiple SNARE complexes are formed in a concurrent manner to maximize their impact to membrane fusion. The SNARE complex is an ideal and important model system for protein complexes working as a team.”
The investigators labeled all the proteins in a SNARE complex with the appropriate dyes. During conventional optical imaging, all the photons coming out from the dyes are detected at once, which makes it impossible to tell one dye from another. Yoon and colleagues defined quantized FRET states, where each state corresponded to a specific number of protein complexes activated at a given moment. They knew the value of the maximum FRET efficiency when all the protein complexes were activated at the same time. Then the researchers measured the total FRET efficiency of the protein complexes and compared this measurement with one of the quantized states. From the maximum value and their measurements, the investigators came up with a ratio that gave information about the number of activated protein complexes out of the total number of possible SNARE complexes.
There are some limitations with the approach. For one, the proteins in the complex have to stay paired long enough for their interaction to be measured. Another limitation is that the dyes have to be precisely and painstakingly attached to the proteins at proper locations to obtain the maximum number of photons out of each interaction.
