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. 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 Resolving Biomolecular Interactions 50 Å

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

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

8. Emission HEAT Absorption E distance = far

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

10. Absorption E Emission F örster R esonance E nergy T ransfer F örster R esonance E nergy T ransfer 10–100 Å DonorAcceptor

11. Key Features of Förster Resonance Energy Transfer: Resonance condition must be met–relaxation energy of donor must approximate excitation energy of acceptor. Choose your FRET pairs wisely! 50 Å λ em = 5210 Å!!! (521 nm) Non-radiative energy transfer—does not involve emission and reabsorption. Distance dependent as 1/r 6, functional range between 10–100 Å. Close range! 50 Å 500 Å

12. TASTE THE FORMATIVE ASSESSMENT

13. http://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. 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 Resolving Biomolecular Interactions 50 Å

16. excite emit excite FRET!

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 Rhodamine B 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?