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Beating detectors at their own game: How to catch a fast molecule in action

Cartoon of single-molecule fluorescence resonance energy transfer (FRET) experiments on bio-molecules called Holliday junctions (HJs). Upon exciting the HJ with blue light, the HJ either emits red or green light. Changes in shapes of molecules result in green light (top right) or red light (bottom right) being emitted.

April 22, 2021

At the small scale at which molecules exist, everything is in motion. Jittering molecules move in every direction by random collisions amongst themselves driven by an effect, studied by Einstein, among others, called Brownian motion.

These motions include changes in distances between molecules but also stretching and contractions of individual molecules. When it comes to large biological molecules, these internal changes in shapes of molecules are referred to as conformational motion.

Conformational motion is critical as it affects how biomolecules interact and describing it provides key insights into understanding biological processes within living systems.

Conformational motion in large biomolecules is often probed by a tool called fluorescence resonance energy transfer (FRET). FRET can be used as a kind of molecular ruler to measure how the distance between two parts of a molecule changes as it undergoes conformational change. Using this technique, a biomolecule is engineered such that it emits green light when two points of interest in the molecule are far apart, and emits red light when two points approach each other as a result of a change in conformation.

When this experiment is performed at the single molecule level, light changes are observed in bursts from individual molecules revealing timescales over which the molecule is changing shape. Measuring rapid changes, as they happen in real time, is difficult with current detectors as these would need to capture images faster than the typical time over which conformational motion occurs. As current detectors can be slower, light bursts appear as mixtures of red and green light, hiding molecular complexity, much like the blurring of a picture of a car speeding on the highway would appear when taken on a cellphone camera. Yet the blur contains useful information!

Now, in a Cell Reports Physical Science article, “Extraction of rapid kinetics from smFRET measurements using integrative detectors,” published this week, Arizona State University researcher Steve Pressé and co-authors report a new mathematical method to extract changes in biomolecular shapes that occur on timescales that exceed detector exposure times.

“So far we have limited ourselves to looking at slow events at the molecular scale," said Pressé, who holds appointments with the Department of Physics, the Center for Biological Physics and the School of Molecular Sciences. "Not because they are interesting to biology but because of how our detectors work. Now, our findings broaden the types of processes we can investigate and expand our ability to peer into increasingly faster events relevant to life.”

“This technique opens up a new window for studying fundamental processes,” said Patricia Rankin, chair and professor of the Department of Physics. “It will give ASU researchers a chance to probe the mechanisms of life and gain new, valuable insights.”

Pressé’s team includes Zeliha Kilic, a postdoctoral associate at the ASU Center for Biological Physics, and Ioannis Sgouralis, an assistant professor at the University of Tennessee, Knoxville’s Department of Mathematics. Experimental collaborators and co-authors of the paper include Tahei Tahara, chief scientist at RIKEN Molecular Spectroscopy Laboratory, also affiliated with RIKEN Ultrafast Spectroscopy Research Team, and his team members Wooseok Heo and Kunihiko Ishii.

"Molecular biology in cells is characterized by many complexities that can't be captured by traditional molecular measurement methods with limited resolutions, so we need new methods to have any hope of properly describing biochemistry in living cells," said Ian Gould, interim director of the School of Molecular Sciences. "The techniques and methodologies being developed in the Pressé lab are expanding the range of useful molecular measurements to the point that they can potentially open up entirely new areas of molecular-level research in cellular systems."

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