The acceptor on the other hand has a different absorption spectrum than the donor. So it won’t absorb the original light. The “only” way it can fluoresce is if it gets the energy directly from the donor. So the acceptor won’t emit light in the absence of the donor. Once it gets energy from the donor, it can emit light (we call this sensitized emission) and this light will be at a different wavelength than the light emitted by the donor (but the same as if it absorbed energy directly from a photon) You can also have acceptors that take in the light but don’t give off light they just release the energy as heat, etc. So you can measure quenching - look at presence or absence of fluorescence versus amounts of fluorescence at 1 wavelength vs the other. In undergrad, I researched a metallopeptidase (a peptide-cutter). To study its cutting activity I used peptides (short amino acid (protein letter) chains) with a donor fluorophore at one end and an acceptor at the other end. The acceptor wasn’t a fluorophore, so it didn’t give off light of its own - it absorbed energy from the 1st but gave that energy off non-radiatively. We call this sort of molecule a quencher. So if FRET occurred, you couldn’t see anything. And the only way FRET could occur is if the peptide got cut because then the fluorophore was freed from the quencher and could shine. There are lots of uses of FRET - you can see if molecules interact (e.g. tag 2 proteins with FRET partners), whether molecules change shape (e.g. tag 2 ends of the same protein with FRET partners), etc. For cell-based assays, you can use molecular cloning to add genes for fluorescent proteins onto the end of the gene for the protein you want to be labeled -> when that gene is made it will have the fluorophore attached. Common fluorescent proteins used for this are optimized versions of CFP and YFP (which are versions of GFP) For test-tube work you can also use small molecule FRET partners like Cy3 and Cy5, which are frequently used to label DNA or RNA. more on GFP: bit.ly/gfpfunscience more on FRET & fluorescence: bit.ly/fretandfluorescence & bit.ly/fluorescentstains Paper examples: * Cecon, E., Burridge, M., Cao, L., Carter, L., Ravichandran, R., Dam, J., & Jockers, R. (2022). SARS-COV-2 spike binding to ACE2 in living cells monitored by TR-FRET. Cell chemical biology, 29(1), 74-83.e4. doi.org/10.1016/j.chembiol.2021.06.008 * Yang, Z., Han, Y., Ding, S., Shi, W., Zhou, T., Finzi, A., Kwong, P. D., Mothes, W., & Lu, M. (2021). SARS-CoV-2 Variants Increase Kinetic Stability of Open Spike Conformations as an Evolutionary Strategy. mBio, 13(1), e0322721. doi.org/10.1128/mbio.03227-21  * more on Spike: bit.ly/coronavirusspike FRET resources: * Bajar, B. T., Wang, E. S., Zhang, S., Lin, M. Z., & Chu, J. (2016). A Guide to Fluorescent Protein FRET Pairs. Sensors (Basel, Switzerland), 16(9), 1488. doi.org/10.3390/s16091488 * Benoit Giquel, Addgene, Tips for using FRET in your experiments blog.addgene.org/tips-for-using-fret-in-your-experiments * Cole N. B. (2013). Site-specific protein labeling with SNAP-tags. Current protocols in protein science, 73, 30.1.1-30.1.16. doi.org/10.1002/0471140864.ps3001s73 * FRET Basics and Applications an EAMNET teaching module Timo Zimmermann + Stefan Terjung Advanced Light Microscopy Facility European Molecular Biology Laboratory, Heidelberg www.med.unc.edu/microscopy/wp-content/uploads/sites/742/2018/06/fret-teaching-module.pdf * Jares-Erijman, E., Jovin, T. FRET imaging. Nat Biotechnol 21, 1387-1395 (2003). doi.org/10.1038/nbt896 * Kim H, Ju J, Lee HN, Chun H, Seong J. Genetically Encoded Biosensors Based on Fluorescent Proteins. Sensors. 2021; 21(3):795. doi.org/10.3390/s21030795 * Liu, L., He, F., Yu, Y., & Wang, Y. (2020). Application of FRET Biosensors in Mechanobiology and Mechanopharmacological Screening. Frontiers in bioengineering and biotechnology, 8, 595497. doi.org/10.3389/fbioe.2020.595497 * Nikon, Fundamental Principles of Förster Resonance Energy Transfer (FRET) Microscopy with Fluorescent Proteins www.microscopyu.com/applications/fret/basics-of-fret-microscopy * Paul R Selvin (2016), "[illinois] Physics 598 Lecture 5: Still more FRET," nanohub.org/resources/24577 * Roger Tsien Nobel lecture: www.nobelprize.org/prizes/chemistry/2008/tsien/lecture/ * Rowland, C. E., Brown, C. W., Medintz, I. L., & Delehanty, J. B. (2015). Intracellular FRET-based probes: a review. Methods and applications in fluorescence, 3(4), 042006. doi.org/10.1088/2050-6120/3/4/042006 * Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat Methods 5, 507-516 (2008). doi.org/10.1038/nmeth.1208 * Verma AK, Noumani A, Yadav AK, Solanki PR. FRET Based Biosensor: Principle Applications Recent Advances and Challenges. Diagnostics. 2023; 13(8):1375. doi.org/10.3390/ * Wu, L., , Huang, C., , Emery, B. P., , Sedgwick, A. C., , Bull, S. D., , He, X. P., , Tian, H., , Yoon, J., , Sessler, J. L., , & James, T. D., (2020). Förster resonance energy transfer (FRET)-based small-molecule sensors and imaging agents. Chemical Society reviews, 49(15), 5110-5139. doi.org/10.1039/c9cs00318e more on binding thermodynamics: bit.ly/bindingaffinityavidity ; TH-cam: th-cam.com/video/aErpulsqG1g/w-d-xo.html & measurement methods: th-cam.com/video/82lYx601WKA/w-d-xo.html
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
It is incredible how you have a video for every single thing I need to know about with more than enough coverage and depth. Great job!
Happy to help! Thanks!
The acceptor on the other hand has a different absorption spectrum than the donor. So it won’t absorb the original light. The “only” way it can fluoresce is if it gets the energy directly from the donor.
So the acceptor won’t emit light in the absence of the donor. Once it gets energy from the donor, it can emit light (we call this sensitized emission) and this light will be at a different wavelength than the light emitted by the donor (but the same as if it absorbed energy directly from a photon)
You can also have acceptors that take in the light but don’t give off light they just release the energy as heat, etc. So you can measure quenching - look at presence or absence of fluorescence versus amounts of fluorescence at 1 wavelength vs the other.
In undergrad, I researched a metallopeptidase (a peptide-cutter). To study its cutting activity I used peptides (short amino acid (protein letter) chains) with a donor fluorophore at one end and an acceptor at the other end. The acceptor wasn’t a fluorophore, so it didn’t give off light of its own - it absorbed energy from the 1st but gave that energy off non-radiatively. We call this sort of molecule a quencher. So if FRET occurred, you couldn’t see anything. And the only way FRET could occur is if the peptide got cut because then the fluorophore was freed from the quencher and could shine.
There are lots of uses of FRET - you can see if molecules interact (e.g. tag 2 proteins with FRET partners), whether molecules change shape (e.g. tag 2 ends of the same protein with FRET partners), etc.
For cell-based assays, you can use molecular cloning to add genes for fluorescent proteins onto the end of the gene for the protein you want to be labeled -> when that gene is made it will have the fluorophore attached. Common fluorescent proteins used for this are optimized versions of CFP and YFP (which are versions of GFP)
For test-tube work you can also use small molecule FRET partners like Cy3 and Cy5, which are frequently used to label DNA or RNA.
more on GFP: bit.ly/gfpfunscience
more on FRET & fluorescence: bit.ly/fretandfluorescence & bit.ly/fluorescentstains
Paper examples:
* Cecon, E., Burridge, M., Cao, L., Carter, L., Ravichandran, R., Dam, J., & Jockers, R. (2022). SARS-COV-2 spike binding to ACE2 in living cells monitored by TR-FRET. Cell chemical biology, 29(1), 74-83.e4. doi.org/10.1016/j.chembiol.2021.06.008
* Yang, Z., Han, Y., Ding, S., Shi, W., Zhou, T., Finzi, A., Kwong, P. D., Mothes, W., & Lu, M. (2021). SARS-CoV-2 Variants Increase Kinetic Stability of Open Spike Conformations as an Evolutionary Strategy. mBio, 13(1), e0322721. doi.org/10.1128/mbio.03227-21

* more on Spike: bit.ly/coronavirusspike
FRET resources:
* Bajar, B. T., Wang, E. S., Zhang, S., Lin, M. Z., & Chu, J. (2016). A Guide to Fluorescent Protein FRET Pairs. Sensors (Basel, Switzerland), 16(9), 1488. doi.org/10.3390/s16091488
* Benoit Giquel, Addgene, Tips for using FRET in your experiments blog.addgene.org/tips-for-using-fret-in-your-experiments
* Cole N. B. (2013). Site-specific protein labeling with SNAP-tags. Current protocols in protein science, 73, 30.1.1-30.1.16. doi.org/10.1002/0471140864.ps3001s73
* FRET Basics and Applications an EAMNET teaching module Timo Zimmermann + Stefan Terjung Advanced Light Microscopy Facility European Molecular Biology Laboratory, Heidelberg www.med.unc.edu/microscopy/wp-content/uploads/sites/742/2018/06/fret-teaching-module.pdf
* Jares-Erijman, E., Jovin, T. FRET imaging. Nat Biotechnol 21, 1387-1395 (2003). doi.org/10.1038/nbt896
* Kim H, Ju J, Lee HN, Chun H, Seong J. Genetically Encoded Biosensors Based on Fluorescent Proteins. Sensors. 2021; 21(3):795. doi.org/10.3390/s21030795
* Liu, L., He, F., Yu, Y., & Wang, Y. (2020). Application of FRET Biosensors in Mechanobiology and Mechanopharmacological Screening. Frontiers in bioengineering and biotechnology, 8, 595497. doi.org/10.3389/fbioe.2020.595497
* Nikon, Fundamental Principles of Förster Resonance Energy Transfer (FRET) Microscopy with Fluorescent Proteins www.microscopyu.com/applications/fret/basics-of-fret-microscopy
* Paul R Selvin (2016), "[illinois] Physics 598 Lecture 5: Still more FRET," nanohub.org/resources/24577
* Roger Tsien Nobel lecture: www.nobelprize.org/prizes/chemistry/2008/tsien/lecture/
* Rowland, C. E., Brown, C. W., Medintz, I. L., & Delehanty, J. B. (2015). Intracellular FRET-based probes: a review. Methods and applications in fluorescence, 3(4), 042006. doi.org/10.1088/2050-6120/3/4/042006
* Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat Methods 5, 507-516 (2008). doi.org/10.1038/nmeth.1208
* Verma AK, Noumani A, Yadav AK, Solanki PR. FRET Based Biosensor: Principle Applications Recent Advances and Challenges. Diagnostics. 2023; 13(8):1375. doi.org/10.3390/
* Wu, L., , Huang, C., , Emery, B. P., , Sedgwick, A. C., , Bull, S. D., , He, X. P., , Tian, H., , Yoon, J., , Sessler, J. L., , & James, T. D., (2020). Förster resonance energy transfer (FRET)-based small-molecule sensors and imaging agents. Chemical Society reviews, 49(15), 5110-5139. doi.org/10.1039/c9cs00318e
more on binding thermodynamics: bit.ly/bindingaffinityavidity ; TH-cam: th-cam.com/video/aErpulsqG1g/w-d-xo.html & measurement methods: th-cam.com/video/82lYx601WKA/w-d-xo.html
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
#scicomm #biochemistry #molecularbiology #biology #sciencelife #science #realtimechem
Excellent lecture! Thank you so much for your help!
You're very welcome!
You are such a great teacher !!😍😍🥰🥰
Thank you so much! Glad it helped!