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F örster Resonance Energy Transfer (Chemistry/Biology Interface)

F örster Resonance Energy Transfer (Chemistry/Biology Interface). Michelle, Pauline, Brad, Thane, Hill, Ming Lee, Huiwang Facilitator: Nancy. Context : Upper level undergraduate or intro graduate course/module in chemistry or biology

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F örster Resonance Energy Transfer (Chemistry/Biology Interface)

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  1. Förster Resonance Energy Transfer(Chemistry/Biology Interface) Michelle, Pauline, Brad, Thane, Hill, Ming Lee, Huiwang Facilitator: Nancy

  2. Context: Upper level undergraduate or intro graduate course/module in chemistry or biology Background: Fluorescent labeling of biomolecules, fluorescent proteins, and confocal microscopy

  3. Goals • Appreciate the optical tools used in biology at a molecular level • Understand principles of fluorescence/luminescence • Appreciate the applications of fluorescence/luminescence in biological systems

  4. Learning Outcomes • Explain/define FRET • Interpret a basic FRET experiment • Suggest potential biological experiments that use FRET

  5. Resolving Biomolecular Interactions 50 Å Optical Microsope ~200 nm resolution Organelle level Great for live cells Electron Microscope Sub-nm resolution Molecule level Not suitable for living cells How can we watch molecules interact in living cells? https://www.tedpella.com/mscope_html/22460-10.jpg http://www.fidelitysystems.com/unlinked_DNA_EM_1.JPG http://www.big.ac.cn/jgsz/kyxt/sysmk/200907/W020090728641466094907.jpg

  6. HEAT excited state! = Fluorescein Absorbs: blue Appears: orange Emits: green E Emission Absorption

  7. HEAT = excited state! Rhodamine B Absorbs: green Appears: red Emits: orange E Emission Absorption

  8. HEAT E Emission Absorption distance = far

  9. HEAT HEAT Radiationless energy transfer E Emission Absorption distance = close Donor Acceptor

  10. Förster Resonance Energy Transfer E Emission Absorption 10–100 Å Donor Acceptor

  11. Key Features of FörsterResonance Energy Transfer: • Non-radiative energy transfer—does not involve emission and reabsorption. λem = 5210 Å!!! (521 nm) 50 Å 50 Å 50 Å 50 Å 500 Å • Resonance condition must be met–relaxation energy of donor must approximate excitation energy of acceptor. Choose your FRET pairs wisely! ✘ ✔︎ • Distance dependent as 1/r6, functional range between 10–100 Å. Close range!

  12. TASTE THE FORMATIVE ASSESSMENT

  13. http://www.hohenstein.de/media/image/press_300dpi/03_farb__und_weissmetrik/479_farbmessung_2013/Wellenspektrum_Licht_EN.jpghttp://www.hohenstein.de/media/image/press_300dpi/03_farb__und_weissmetrik/479_farbmessung_2013/Wellenspektrum_Licht_EN.jpg

  14. Report Out • What were the outcomes of your trials? • Did every excitation event result in FRET? • Did every excitation event result in a fluorescence event? • How did group size effect the outcome?

  15. Resolving Biomolecular Interactions 50 Å Optical Microsope ~200 nm resolution Organelle level Great for live cells Electron Microscope Sub-nm resolution Molecule level Not suitable for living cells How can we watch molecules interact in living cells? https://www.tedpella.com/mscope_html/22460-10.jpg http://www.fidelitysystems.com/unlinked_DNA_EM_1.JPG http://www.big.ac.cn/jgsz/kyxt/sysmk/200907/W020090728641466094907.jpg

  16. emit excite excite FRET! emit

  17. Brainstorming Using what you have learned about FRET, suggest a biological question that could be illuminated via a FRET experiment.

  18. Summative Assessment (LOCS) • ________ Amino acid X and Y are thought to be ~1000 Å apart on a protein. FRET could be a useful tool to measure this distance. • _____The wavelength of light is directly proportional to its energy. • _____ For FRET to occur, the absorption spectrum of the acceptor should overlap with the emission spectrum of the donor. • _____ The Förster transfer of energy from a donor to an acceptor involves the emission and reabsorption of a photon. • _____ Emission from the donor is indicative of FRET. • F, F, T, F, F.

  19. Summative Assessment (HOCS) Proteins A and B are membrane bound and labeled with Fluorescein and Rhodamine B, respectively. Your labmate intends to excite his cells at 555 nm and look for the RhodamineB emission at 580 nm as evidence of the complexation of proteins A and B. Assess his experimental design and suggest solutions to any potential problems. Your labmate cuts you off mid-sentence, realizing his error, and adjusts his instrument to observe the fluorescein emission at 521 nm. Is he on the right track?

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