A beginner's guide to this simulation
Proteins are tiny machines that constantly change shape as they do their job. Diffusion-based single-molecule Förster resonance energy transfer (smFRET) experiments detect bursts of fluorescence as individual labelled biomolecules transiently enter the observation volume, providing snapshots of their conformational dynamics.
This simulation models particles diffusing through a confocal volume and stochastically switching between two conformational states with distinct FRET efficiencies. Each state yields different donor and acceptor emission rates, which are plotted in real time at the bottom of the page. As you watch individual particles change shape, you can directly correlate those conformational switches with jumps in the green (donor) and orange (acceptor) count-rate traces, just as you would observe in diffusion-based single-molecule FRET experiments.
Each burst is analysed to calculate an average FRET efficiency, and that value is added as a count to the FRET histogram. Over time, the histogram builds up two peaks, one for each conformation of the protein.
In real experiments, watching proteins in solution undergo these conformational transitions allows researchers to probe their dynamics and understand how these shape changes relate to biological function.
FRET stands for Förster Resonance Energy Transfer. When two fluorescent dyes, called the donor and acceptor, are attached to a protein and brought very close together, energy can jump from the donor to the acceptor. The acceptor then glows instead of the donor.
FRET efficiency (E) runs from 0 to 1. E = 1 means all energy is transferred to the acceptor (dyes very close). E = 0 means no transfer at all (dyes very far apart). In this simulation, the compact state gives E ≈ 0.75 and the extended state gives E ≈ 0.25.
In a typical lab experiment, you would measure millions of molecules at once and only ever see an average. smFRET uses a tiny focused laser beam (the glowing hourglass shape in the middle of the screen, called the confocal volume) so small that usually only one molecule is inside it at any moment. As molecules drift in and out, you catch them one at a time.
Because the protein switches between its compact and extended forms. When many bursts have been recorded, you can see how often the protein is in each state and how fast it switches. This is information that is very hard to obtain any other way.
Particles sets how many molecules are in solution. More molecules means more bursts per second but also a greater chance of two molecules being in the beam at once.
Molecule size changes how big the dumbbells appear on screen.
Diffusion (D) controls how fast the molecules move. Real proteins in water diffuse at around D = 100 µm²/s.
Rates set how fast the protein switches between compact and extended conformations.
FCS mode adds 10 times more molecules, mimicking a higher concentration sample.
Fast simulation speeds up all the physics so the histogram fills in more quickly.
Joel A. Crossley
University of Leeds