A lot of cutting edge research today uses fluorescence-related technologies, such as fluorescence imaging, fluorescence anisotropy/polarisation, or the more popular Förster resonance energy transfer (FRET). While fluorescence imaging refers to a really broad technique that can be used to study anisotropy and FRET, the latter two are physical phenomena that can be used as a proxy for visualizing an underlying biological process such as biomolecular interactions, protein mobility, protein unfolding, membrane fluid dynamics, and so on.
If you are planning a FRET experiment or even just thinking about it for future use, this article will give you a brief understanding of the principles, requirements, and procedures that will help you successfully perform your first FRET experiment.
What is FRET and why should you seriously consider it?
FRET is a physical process by which energy from one chromophore (fluorescent or phosphorescent) molecule is transferred to another molecule that is capable of absorbing that energy (need not be fluorescent) in a non-radiative, distance-dependent fashion. The key concept to keep in mind here is that the process is non-radiative, which means that it does not involve emission of any kind of electromagnetic radiation, such as photons. While understanding this concept does not affect the actual performing of a FRET experiment, it will definitely help people take you more seriously when you can talk about it!
The second key concept, distance dependence, is more important for biologists. When two chromophores get close together with their dipoles aligned in suitable orientations, one chromophore (known as the donor) can transfer its excited state energy to the second chromophore (known as the acceptor) due to transient dipole-dipole coupling. The transfer of energy from donor to acceptor is proportional to the inverse of the sixth power of distance between them. What this means is that for a unit increase in distance, the energy transfer decreases by a power of six. This extreme sensitivity to even small changes in distance ensures that in a biological scenario, two labeled biomolecules can only show FRET if they are closer than 10nm from each other. Using FRET as a proxy, the binding between two biomolecules and the affinity of their interaction in terms of dissociation constants can be calculated.
The advantage of FRET is that this technique can provide spatio-temporal information when combined with fluorescence microscopy. Interactions and their rates can be studied in real time in live cells, including signalling events that happen in time scales of seconds. Since the method is non-invasive, the same cell can be used to study the response of the interaction under different conditions or stimuli.
In addition to these advantages, FRET can be used to study the effect of various post-translational modifications, mutations, domains, metal ion, and cofactor requirements on the interaction between two (chromophore-labeled) biomolecules. In certain innovative experiments, FRET between two domains of a single protein has been used to study the conformational changes of a protein during an event such as ligand binding.
FRET is also compatible with commonly used plate-reader technologies where 96, 384 or even 1,536 interactions can be studied simultaneously. Since the interactions are measured in real time, FRET can even be used in competition assays between two binding partners to see which pair has the greater interaction affinity. These make FRET a powerful tool in high throughput screening assays for drugs or ligands.
If you’re sold on using FRET for your next experiment or project, read Part 2 of this series, which will guide you through the steps required for making FRET happen!
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Vignesh is a molecular biologist working at the Indian Institute of Science, Bangalore, India with a passion for science communication. When he is not playing with his fluorescence microscope, he tries to simplify science through his writing and is actively looking to transition into full time science writer and editor.