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?