FRET Basics and Applications an EAMNET teaching module

FRET Basics and Applications

an EAMNET teaching module

Timo Zimmermann + Stefan Terjung

Advanced Light Microscopy Facility

European Molecular Biology Laboratory, Heidelberg

http://www.embl.de/almf/

http://www.embl.de/eamnet/

Overview

1) Fluorescence Resonance Energy Transfer Basics

2) Confocal FRET detection techniques

3) FRET and fluorescent proteins

4) A new GFP FRET pair with increased efficiency

The resolving power of light microscopes is limited to distances of hundreds of nanometers (<organelles).

Fluorescence Resonance Energy Transfer (FRET) allows the detection of molecule-molecule interactions in the nanometer range with light microscopy.

FRET is sometimes also called Förster Resonance Energy Transfer, as Förster was the first who published quantitative theory of molecular resonance energy transfer (Förster 1946, Förster 1948).

1 nm 10-9_m 1 µm 10-6_m 1 mm 10-3_m 1 cm 10-2_m 1 m 1 Å 10-10_m

Cells Worm Housefly

Organelles Human

FRET

light microscopy resolution limit

Fluorescence Resonance Energy transfer (FRET)

FRET is a non-radiative transfer of energy from an excited donor molecule to a suitable acceptor molecule in close proximity.

Wouters et al. (2001), TICB 11/5

Fluorescence Resonance Energy Transfer

In the case of FRET, excitation of the donor fluorophore results not only in donor emission, but partially also in emission

characteristic for the acceptor fluorophore.

Dependence on distance and spectral overlap

The efficiency of energy transfer strongly depends on the distance between the donor

acceptor molecules and on overlap of the donor molecule emission and acceptor molecule excitation spectra Î high specificity.

FRET efficiency is depends on molecule distance

and

The FRET efficiency depends on the distance between the two interacting molecules. At the distance of the Förster radius R₀ between the molecules, the FRET efficiency is 50%. The typical R₀ is around 3 nm.

Donor/Acceptor Pairs

Examples for common FRET Donor/Acceptor pairs:

Donor (Em.)

Acceptor (Exc.)

FITC (520 nm)

TRITC (550 nm)

Cy3 (566 nm)

Cy5 (649 nm)

EGFP(508 nm)

Cy3 (554 nm)

CFP (477 nm)

YFP (514 nm)

EGFP (508 nm)

YFP (514 nm)

FRET detection methods

A variety of FRET detection methods exist for light microscopy

Acceptor photobleaching

Donor photobleaching

Ratio imaging

Sensitized emission

FRET detection methods

The detection methods have different properties and are suited to different samples

Detection of changes:

Acceptor photobleaching

Donor photobleaching

Information self-contained:

Ratio imaging

Sensitized emission

=> fixed samples

=> in vivo

Fluorescence lifetime measurements

Acceptor Photobleaching

Experimental steps of acceptor photobleaching measurements

In acceptor photobleaching, the acceptor molecule of the FRET pair is bleached, resulting in a brightening (unquenching) of the donor fluorescence.

Prebleach Image Bleaching Postbleach Image Median Filtering Subtraction: Postbleach – Prebleach Division: Subtraction/ Postbleach Zoom 4x Original Zoom GFP GFP Cy3 Cy3 488 488 488 543 543 543

An apparent FRET efficiency (product of the efficiency of the FRET pair and the amount of interacting donor) can be calculated

Acceptor photobleaching

Shift Correction by Cross-Correlation helps avoiding edge artifacts in the comparison of pre- and postbleach images.

Edge artifacts

Donor photobleaching

FRET decreases donor fluorescence lifetime => decreased likeliness of bleaching

=> decreased bleaching rate

Fluorescence lifetime

The bleaching rate of the donor fluorophore is affected by FRET. Measuring the bleaching of the donor in the presence/absence of acceptor is a possibility to detect FRET.

An apparent FRET efficiency (product of the efficiency of the FRET pair and the amount of interacting donor) can be calculated.

However: Quantitation is problematic due to direct and indirect bleaching of acceptor

Overview

1) Fluorescence Resonance Energy Transfer Basics

2) Confocal FRET detection techniques

3) FRET and fluorescent proteins

4) A new GFP FRET pair with increased efficiency

FRET and Fluorescent Proteins (FPs)

Protein-Protein Interactions:

- FRET between an FP and a dye

- FRET between FPs

Cameleons:

In vivo measurements of physiological

changes (ratio imaging)

GFP-Protein GFP-Protein

P

d>Ro

d<Ro

Measurement of Protein Phosphorylation by FRET

Cy3 anti-P-Thyr

Application example: An acceptor-labelled antibody against a phosphorylated residue can be used to detect the phosphorylation status of a GFP-fusion

protein by FRET

Phosphorylated

Not Phosphorylated

Verveer, et al. 2000

Acceptor photobleaching

Receptor phosphorylation after EGF-Stimulation

0 min 2 min 5 min

CFP/YFP

The combination of cyan and yellow fluorescent protein is the most commonly used fluorescent protein FRET pair

Fluorescence Resonance Energy Transfer

Cameleon Tandem constructs

CFP YFP

In vivo CFP/YFP cameleon measurements

Measurements caried out on the Leica SP2 AOBS at 405 nm excitation:

2 µM Ionomycin + 20mM CaCl2

Histamine EGTA

Cross-talk and cross-excitation of two fluorophores is an intrinsic problem of multichannel

measurements and is also present in FRET measurements

Channel 1: 460-500 nm Channel 2: 460-500 nm RGB Overlay CFP only YFP only CFP+ YFP

Sensitized emission detection

Ratiometric imaging can only be done in samples with a fixed stochiometry of donor and acceptor (e.g. Cameleons) D A A S. Terjung + T. Zimmermann D

A In samples with variable

stochiometries, the detected acceptor fluorescence has to be corrected for emission cross-talk and for cross-excitation

A D A A D A _D A A

Sensitized emission detection

Predetermined factors with pure samples of donor and acceptor: Donor cross-talk : R_D Acceptor cross-excitation: R_E Donor channel Donor excitation FD Acceptor channel Donor excitation FDA Acceptor channel Acceptor excitation FA corr Donor cross-talk correction Acceptor cross-excitation correction Required images: FDA _corr/FA

=>

Overview

1) Fluorescence Resonance Energy Transfer Basics

2) Confocal FRET detection techniques

3) FRET and fluorescent proteins

4) A new GFP FRET pair with increased efficiency

CFP/YFP

Cyan and yellow fluorescent protein is the most commonly used fluorescent protein FRET pair

Requirements for a good FRET pair

-Maximal overlap of donor emission and

acceptor excitation

-Minimal direct excitation of the acceptor at the

excitation maximum of the donor

Spectral overlap of FRET Pairs

The spectral overlap of donor emission and acceptor excitation is only partial for CFP/YFP and much better for GFP/YFP pairs

Requirements for a good FRET pair

-Maximal overlap of donor emission and

acceptor excitation

-Minimal direct excitation of the acceptor at the

excitation maximum of the donor

Different Cross-Excitation of FRET Pairs

Using a suitable laser excitation for CFP, YFP is directly excited significantly (=> high background signal)

GFP2 is excitable around 400 nm, where YFP is almost not excitable (=> low background signal)

Comparison of CFP/YFP and

GFP2/YFP FRET pairs

CFP YFP exc. 405/458 nm glycine linker GFP2 YFP exc. 405 nm glycine linker S. Terjung + T. Zimmermann

Acceptor photobleaching

Comparison of CFP and GFP2 in the same construct

Before After

CFP-YFP: FRET efficiency 20% GFP2-YFP: FRET efficiency 30% => 50% increase

Improved FRET Efficiency significantly improves Detection

Whereas the differences between FRET pairs are not significant at high transfer efficiencies, a more efficient FRET pair significantly improves the detectable FRET interaction in cases of low FRET efficiency.

Sensitized emission of GFP2-YFP FRET pairs

YFP (sensitized emission) YFP (direct excitation)

GFP2+YFP Coexpression

GFP2-YFP linked

Comparison of CFP/YFP and GFP2/YFP

FRET pairs

- 32% increased overlap of donor emission and acceptor

excitation

- Higher absorbance and quantum efficiency of the donor

- Higher Foerster Radius (approx. 5.5 nm)

- Increased FRET efficiency, especially at longer distances

- Suitable for donor photobleaching

- However:

Linear unmixing of the strongly overlapping

emission signals required

ALMF: Rainer Pepperkok

Jens Rietdorf

Stefan Terjung

GFP2/YFP project: Andreas Girod

Virginie Georget

Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2 -YFP FRET pair T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, R. Pepperkok, FEBS Letters 531 (2002)245 -249

http://www.embl.de/almf/

http://www.embl.de/eamnet/

Literature

• T. Förster (1946): Naturwissenschaften 6, 166 • T. Förster (1948): Ann. Phys. (Leipzig) 2, 55

• A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura and R. Y. Tsien (1997): Nature 388, 882-887.

• B.A. Pollok and R. Heim (1999): Trends in Cell Biology 9, 57-60.

• P.J. Verveer, F.S. Wouters, A.R. Reynolds, P.I. Bastiaens (2000): Science 290, 1567-1570

• F.S. Wouters, P. J. Verveer and P. I. H. Bastiaens (2001): Trends Cell Biol 11, 203-211. • T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, R. Pepperkok (2002): FEBS Letters

531, 245 -249