Coupling Single-Molecule FRET and Microfluidic Mixing
Scientists at The Scripps Research Institute and the University of California, San Diego (UCSD) recently reported on a new technique that enables detection in less than 0.001 seconds of transiently folded single-molecule structures from a class of rapidly shape-shifting molecules known as intrinsically disordered proteins (IDPs). The method, described in Nature Methods, also permits new types of observations of short-lived protein complexes and allows analysis of IDPs associated with human neurodegenerative disorders, including the α-synuclein protein associated with Alziehmer and Parkinson diseases.
Unlike proteins with a well-defined 3-D structure that determines their cellular functions, IDPs have long stretches of sequence that are relatively devoid of structure. These protein systems are very prevalent in genomes, especially in more evolved organisms. The physics of these systems may play a key role in many cellular functions including signaling, scaffolding, gating, and timing, according to Ashok A. Deniz, Ph.D., associate professor, department of molecular biology, California campus of Scripps.
To address analyses of these proteins, Dr. Deniz and co-researchers Alex Groisma, Ph.D., associate professor of physics at UCSD, and Yann Gambin, Ph.D., of Scripps, combined and improved upon two established experimental methods: single-molecule Förster Resonance energy transfer (smFRET) and microfluidic mixing.
“Fast laminar microfluidic flows have been used for fast mixing, but under these conditions, the molecules go by the detectors too fast to collect enough photons for good signal-to-noise for smFRET measurements,” Dr. Deniz told GEN.
“Hence, previous smFRET measurements used slower flows resulting in slower mixing. In our device, by combining fast mixing in fast flow with rapid deceleration just prior to detection, we could then collect enough photons for smFRET analysis very soon after a change in conditions.”
Dr. Deniz told GEN that understanding the structural properties and complicated dynamics of intrinsically disordered proteins is difficult with many conventional ensemble experiments that average properties of millions of molecules. Commenting on the microfluidics used in his approach, he said, “We reported a method in which you can look at changes in structural distributions following a rapid change in conditions—in the case of α-synuclein, the presence of a binding partner—to see what happens to the distribution as a function of time.”
“We have pushed the time resolution to below a millisecond for microfluidic single-molecule FRET under nonequilibrium conditions. And not only can we change the conditions surrounding the protein, but we can also dilute the protein solution rapidly, which is very useful for looking at relatively weak complexes that would normally dissociate under single protein conditions. Now we can watch them for a while before they dissociate.”
Leveraging a similar technique to Dr. Deniz’, physicists working at UC Santa Barbara (UCSB) in collaboration with colleagues at the University of Zurich made the first subsecond, single-molecule measurements of a chaperonin protein. Their study, published in the June 2010 issue of the Proceedings of the National Academy of Sciences, used a microfabricated fluid mixing device built at UCSB’s nanofabrication facility.
Chaperonins comprise a group of proteins that help other proteins fold correctly and prevent protein aggregation. GroEL, one member of this protein family, works with GroES to encapsulate a substrate protein, isolating it as it folds in the cell. Inside the fluid mixing device the scientists followed the folding of the protein rhodanase inside the GroEL/GroES chaperonin molecular cavity over a time range from milliseconds to hours.