The key innovation was to combine rapid mixing in a high-speed flow with single-molecule detection in a slow flow.

An international team of researchers have developed a technique that allows detection in less than 0.001 second of previously unknown details of transiently folded single-molecule structures formed from intrinsically disordered proteins. The method also enables observations of short-lived protein complexes.

Details of the technique are reported in “Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing,” which appears in an advanced, online issue of Nature Methods.

The scientists examined alpha-synuclein—a protein linked to Parkinson and Alzheimer diseases. They expect discoveries made as a result of applying their new technique to this protein will help in the study of plaque formation connected with neurodegenerative disorders.

Known techniques to determine protein structures are often designed for ordered proteins, and detection of transient shapes in structurally heterogenous proteins such as alpha-synuclein has been difficult, the researchers note. To overcome the shortcomings of current techniques, the investigators combined two experimental methods: single-molecule detection via Förster Resonance Energy Transfer (smFRET) in a slow flow and rapid microfluidic mixing in a high-speed laminar flow. While high-speed microfluidic mixing has been used previously to rapidly initiate protein-folding reactions, the researchers note that most observations have been made on a bulk rather than single-molecule level.

The researchers mixed alpha-synuclein protein in an aqueous solution with sodium dodecyl sulfate (SDS). The mixing took place inside a small chip housing several hollow channels, including a main channel for proteins. This channel connects to other inlets or outlets.

Two inlets were used to funnel buffer and SDS into the central stream, thus focusing the central protein stream into a narrow, fast-moving lane and allowing a rapid switch into a solution containing SDS. Further along the channel, two outlets forced much of the protein to exit, causing the speed of the remaining central part of the stream to abruptly decrease. Protein molecules in the slower, focused protein stream are detected by smFRET.

The rapid slowing was a critical new element in the method, providing just enough time for scientists to examine individual slower-moving proteins as they passed by the detector, the researchers remark.

Alpha-synuclein was introduced at an inlet to negatively charged SDS, which prompted it to fold. Combined with the rapid mixing, the fluorescence from the dye tags—which had been placed far apart on alpha-synuclein—revealed previously unknown details of transiently folded structures of alpha-synuclein, observed in the submillisecond timeframe.

Prior to this work, the equilibrium state of alpha-synuclein in the SDS-containing solution was known to be an extended helix called the F state. This ordered structure exists in the presence of the negatively charged biological membrane or SDS.

“So the question was: ‘Do we go directly from the disordered protein to that F state?’” explains Ashok Deniz, Ph.D., a Scripps Research associate professor and co-leader of the study.

“And the answer from our experiments was, ‘No.’ We visit an intermediate structure, which has a similar FRET efficiency to what was previously observed to be a helix-kink-helix (I state), like a coil with a kink that bends the coil into a U-shape instead of a straight coil.

“Surprisingly, even this initial transition is complex and provides us views of how the protein shape changes soon after binding to its partner molecules. What this means is that, as conditions in the cell are dynamic, these new states might give us many more points of regulation of alpha-synuclein.”

Next in the lab’s research, Dr. Deniz plans to examine whether different alpha-synuclein structures aggregate differently and how they couple to function. His group will also address what triggers the aggregation, the roles of these aggregates, and what kinds of structures will be detected for alpha-synuclein interacting with other protein, lipid, and small-molecule partners.

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