f örster resonance energy transfer (chemistry/biology interface)
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Förster Resonance Energy Transfer(Chemistry/Biology Interface)
Michelle, Pauline, Brad, Thane, Hill, Ming Lee, HuiwangFacilitator: 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 resolutionOrganelle levelGreat for live cells
Electron MicroscopeSub-nm resolution
Molecule levelNot suitable for living cells
How can we watch molecules interact in
living cells?
https://www.tedpella.com/mscope_html/22460-10.jpghttp://www.fidelitysystems.com/unlinked_DNA_EM_1.JPGhttp://www.big.ac.cn/jgsz/kyxt/sysmk/200907/W020090728641466094907.jpg
Resolving Biomolecular Interactions
50 Å
Emiss
ion
HEAT
Absorption
E
FluoresceinAbsorbs: blue
Appears: orangeEmits: green
=excited state!
Emiss
ion
HEAT
Absorption
ERhodamine B
Absorbs: greenAppears: redEmits: orange
=excited state!
Emiss
ion
HEAT
Absorption
E
distance = far
HEAT
Absorption
E
Emiss
ion
HEAT
Radiationless energy transfer
distance = close
Donor Acceptor
Absorption
E
Emiss
ion
FörsterResonance
EnergyTransfer
10–100 Å
Donor Acceptor
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 λem = 5210 Å!!! (521 nm)
• Non-radiative energy transfer—does not involve emission and reabsorption.
• Distance dependent as 1/r6, functional range between 10–100 Å. Close range!
50 Å50 Å
✔︎ ✘
50 Å 500 Å
TASTE THE FORMATIVE ASSESSMENT
http://www.hohenstein.de/media/image/press_300dpi/03_farb__und_weissmetrik/479_farbmessung_2013/Wellenspektrum_Licht_EN.jpg
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 resolutionOrganelle levelGreat for live cells
Electron MicroscopeSub-nm resolution
Molecule levelNot suitable for living cells
How can we watch molecules interact in
living cells?
https://www.tedpella.com/mscope_html/22460-10.jpghttp://www.fidelitysystems.com/unlinked_DNA_EM_1.JPGhttp://www.big.ac.cn/jgsz/kyxt/sysmk/200907/W020090728641466094907.jpg
Resolving Biomolecular Interactions
50 Å
excite
emit
emit
excit
e
FRET!
BrainstormingUsing 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?
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