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

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Presentation transcript:

Förster 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 Background: Fluorescent labeling of biomolecules, fluorescent proteins, and confocal microscopy

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

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

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? Resolving Biomolecular Interactions 50 Å

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

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

Emission HEAT Absorption E distance = far

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

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

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

TASTE THE FORMATIVE ASSESSMENT

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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?

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? Resolving Biomolecular Interactions 50 Å

excite emit excite FRET!

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

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.

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?