FocalPlane features… Samrat Mukhopadhyay with PicoQuant

In case you missed ‘FocalPlane features…’, here is the recording of the webinar given by Samrat Mukhopadhyay on ‘A deep dive into biomolecular condensates using single-molecule FRET’, which we co-hosted with PicoQuant. Unfortunately, our automatic recording failed and we missed the first few minutes of Samrat’s webinar. We’d like to apologise to Samrat and our viewers for this mistake.

To find out more about the PicoQuant MicroTime 200, which was used extensively in Samrat’s research, check out the accompanying post by Maria Loidolt-Krüger from PicoQuant.

Abstract: Cells contain membrane-enclosed organelles that compartmentalize cellular constituents and regulate biochemistry. A growing body of fascinating research now reveals that there is also an alternative mechanism of spatiotemporally-controlled intracellular compartmentalization and organization via liquid-liquid phase separation of proteins and nucleic acids into noncanonical membrane-less organelles. These functional liquid-like biomolecular condensates can undergo aberrant irreversible phase transitions into gel-like or solid-like amyloid aggregates associated with a range of debilitating human diseases. In this talk, I will describe that single-molecule FRET studies offer an incredibly powerful approach to illuminating the exquisite inner workings of phase-separated biomolecular condensates. These studies allow us to dissect the coexisting conformational states and unmask the crucial molecular events that are otherwise skewed in conventional ensemble-averaged experiments. Our findings provide broad mechanistic underpinnings involving an intriguing interplay of sequence-encoded dynamically-controlled making and breaking of weak interactions that govern biological phase transitions involved in cell physiology and disease.

Extra Q&As

Thank you, Professor. In the probability distribution of FRET efficiency, how do you explain the FRET efficiency having value greater than 100? There are some regions having value > 100.

This is due to the fact we have photon shot noise that broadens the histogram on both sides. Shot noise arises due to a low number of photon counts per burst in single-molecule detection (this issue is more pronounced in the droplets where we have experimental limitations in collecting a large number of photons per burst).

Wouldn’t intermolecular interactions results in FRET as well? Since all the molecules have donor and acceptor fluorophores, why is it that fret probability decreases in phase condensates?

We use a sparse amount of dual FRET-labeled protein (picomolar) in the midst of a large amount of unlabeled protein (micromolar) minimizing the contribution from intermolecular FRET. There is practically no contribution from intermolecular FRET.

Fascinating! Very naïve question, how many dyes can be used at max for FRET or smFRET to understand protein complexes?

One can, in principle, do a four-color FRET. However, due to practical difficulties, only three-color FRET (one donor and two acceptors) experiments are currently possible.

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