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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 rster resonance energy transfer chemistry biology interface

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
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
Learning Outcomes

  • Explain/define FRET

  • Interpret a basic FRET experiment

  • Suggest potential biological experiments that use FRET


Resolving biomolecular interactions
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


HEAT

excited state!

=

Fluorescein

Absorbs: blue

Appears: orange

Emits: green

E

Emission

Absorption


HEAT

=

excited state!

Rhodamine B

Absorbs: green

Appears: red

Emits: orange

E

Emission

Absorption


HEAT

E

Emission

Absorption

distance = far


HEAT

HEAT

Radiationless energy transfer

E

Emission

Absorption

distance = close

Donor

Acceptor


Förster

Resonance

Energy

Transfer

E

Emission

Absorption

10–100 Å

Donor

Acceptor


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!



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


Report out
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?


Resolving biomolecular interactions1
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


emit

excite

excite

FRET!

emit


Brainstorming
Brainstorming

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


Summative assessment locs
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
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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