Fluorescence Resonance Energy Transfer using Color Variants of GFP

Dale W. Hailey, Trisha N. Davis and Eric G. D. Muller*

Department of Biochemistry, University of Washington

Seattle, Washington 98195

*Corresponding author:

Department of Biochemistry

University of Washington

Box 357350

Seattle, Washington 98195

FAX: 206-685-1792

Email: emuller@u.washington.edu

Running title: FRET in Yeast

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Introduction

The recent discovery that the human genome encodes only 5 times the number of genes

found in the yeast genome suggests that the fundamental cellular biology of a human cell is not

that much more complex than the cell biology of a yeast cell. As a step towards a greater

understanding of yeast cell biology, the Yeast Resource Center (YRC) at the University of

Washington (http://depts.washington.edu/~yeastrc/) integrates four technologies to study the

components, the function, the structure and the dynamic modifications of protein complexes in

yeast. Those technologies are mass spectrometry, two-hybrid analyses, optical sectioning

microscopy, and protein structure prediction. The goal is to identify not only the constituents of

protein complexes, but also to determine how protein complexes interact to form networks of

interconnected pathways.

Protein localization in live cells

The function of a protein is intimately associated with its location in the cell.

Fluorescence microscopy of live cells expressing proteins fused to the green fluorescent protein

(GFP) allows the visualization of proteins and protein complexes in their natural context (1). In

yeast the replacement of a protein with a GFP-protein fusion is easy to accomplish (2, 3). If the

target protein is essential for viability, the function of the GFP-protein fusion can be tested for its

ability to support growth. We have tagged 17 essential genes and found that only one fusion

protein could not support growth and two conferred slow growth. In addition, driving gene

expression from the native promoter insures that the GFP-protein fusion does not flood the cell

with more protein than the cell can properly assimilate. Given these simple precautions the

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localization of the GFP fusion will likely reflect the natural distribution of the protein in the cell

and yield important information on protein function.

In principle several proteins could be tagged and resolved simultaneously using the

different color variants of GFP and dsRed (1, 4). However we have found that proteins tagged

with either dsRed or the blue fluorescent protein yield weak or undetectable fluorescent signals

in yeast (unpublished results, see also (5) ). Thus in practice only the two color variants cyan

fluorescent protein (CFP) and yellow fluorescent protein (YFP) (1) are available for

simultaneous expression and co-localization studies in yeast. A full description of the plasmids

and methods for generating CFP and YFP fusions is described below. The reader is referred to

several recent papers which provide excellent overviews on fluorescent microscopy in yeast

using GFP (6, 7, 8).

Fluorescence microscopy with modern hardware and software achieves spatial resolution

close to the theoretical limits of optical resolution, 200 – 300 nm. Thus proteins within this

distance of each other will co-localize. For example the yeast spindle pole body, a structure that

is embedded in the nuclear envelope and nucleates microtubules, is about 200 nm across (9). The

fluorescence intensity from any two proteins of the spindle pole body tagged with CFP and YFP

coincide; the distance between them cannot be measured directly.

Localization to defined subcellular structures at a resolution of 200-300 nm is often

sufficient to assign a broad function to many proteins. For example mapping proteins to the

spindle pole body implies a shared role in microtubule organization, whereas a bud tip

localization implies a role in polarized growth. However some localizations, such as the

cytoplasm, are not very informative. In addition, to probe deeper into the mechanism of action of

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a protein complex requires a deeper understanding of molecular structure that only comes at

higher spatial resolution.

Fluorescence resonance energy transfer

Fluorescence resonance energy transfer, or FRET, is the exchange of energy between

two fluorescent molecules, one acting as a donor and the other acting as an acceptor. FRET

occurs only if the acceptor is within a radial distance of 10 nm (100 Å) from the donor.

According to theory proposed by Förster and verified experimentally in model systems

(reviewed in (10)) the efficiency of energy transfer depends on the inverse sixth power of the

distance between the donor and acceptor. Thus the detection of FRET is a very sensitive measure

of the proximity of two fluorophores. When applied to fluorescence microscopy, a FRET signal

narrows the predicted distance between two fluorophores from within 200 nm to as little as 10

nm. In effect FRET increases the spatial resolution of fluorescence microscopy by 20-fold.

FRET transfer efficiency depends on several factors in addition to distance. An

orientation factor, K2, expresses the dependence on the angular relationship between the donor

and acceptor transition moments. K2 can have a value from 0 to 4, and could in theory have a

large impact on FRET efficiency. However in biological systems K2 is usually considered to be

2/3 because of the rotational freedom of the fluorophores (1, 10, 11).

Besides these geometric variables FRET efficiency also depends on several spectroscopic

variables. These include the spectral overlap of the emission spectrum of the donor with the

excitation spectrum of the acceptor, the rate constant for fluorescence emission of the donor, the

quantum yield of fluorescence of the donor in the absence of acceptor, and to a minor extent the

refractive index of the medium.

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For the CFP donor/YFP acceptor FRET pair, the distance at which the energy transfer

efficiency is 50% was calculated to be 50 Å (11). This value assumes a K2 of 2/3. It should be

noted that the shortest possible distance between the CFP and YFP fluorophores is 30 Å since the

fluorophores are buried 15 Å beneath the surface of the proteins. Thus the efficiency of transfer

can never be 100%. To enable FRET to occur at efficiencies greater than 25%, proteins tagged

with CFP and YFP must not only be in contact, but the protein-protein interaction must bring

CFP and YFP together to within 30 Å of each other, again assuming a K2 of 2/3.

Practical Approach to FRET Microscopy in Yeast

This paper is meant as a step by step introduction to the use of FRET to identify and

localize interacting proteins in yeast using CFP and YFP protein fusions. Several recent reviews

on FRET microscopy using GFP variants are excellent sources for further information (11, 12,

13, 14).

Equipment

High performance microscopes are required for detecting FRET in yeast. The small size

of yeast and the faint signals from the fluorescent proteins place exacting demands on the optics

and image acquisition. We use a Zeiss Axiovert S-100 TV incorporated into the DeltaVision®

Restoration Microscopy System. Our primary objective is the 100X oil immersion Plan-

Apochromat® with a numerical aperture of 1.4. To check adjustments, calibrations and

sensitivity on our microscope we employ the InSpeck™ Green and FocalCheck™ fluorescent

microbeads from Molecular Probes as external standards.

Images are acquired on a cooled charge-coupled device camera (Roper Scientific,

Photometrics Quantix camera with a Kodak 1401 chip). Again the dim signals argue for a cooled

CCD camera with low electronic noise, high quantum efficiency in the green region of the

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spectrum, and fast readout speeds. The binning features in a camera are also important. Binning

combines charges from adjacent pixels in a CCD during readout. We routinely increase the

signal to noise ratio by 2x2 binning, which also permits shorter exposure times and reduces

fluorophore bleaching.

Our optical filter sets are from Omega Optical. For CFP, we use 440AF21 for excitation

and 480AF30 for emission. For YFP we use 500AF25 for excitation and 545AF35 for emission.

To observe FRET, the sample is excited with the 440AF21 excitation filter and emission is

examined with the 545 AF35 emission filter. We use a dual pass dichroic mirror, 465-

543DBDR, which decreases the shoulder of the CFP emission into the YFP channel in FRET

experiments. The dual pass dichroic mirror is required if YFP fluorescence is measured during

the FRET experiment as suggested below. The optical filters are placed in filter wheels and

rotated into position through the software that operates the microscope.

Several software packages are available for image analysis and manipulation. We use softWoRx

from Applied Precision, but MetaMorph (Universal Imaging Corp.), ISee (Inovision Corp.) and NIH

Image are also commonly used for image processing.

Technological advances steadily improve the capabilities of fluorescence microscopy. Therefore

this description of equipment is meant only as a guide and not as a shopping list. In particular,

improvements in the sensitivity and speed of cameras and the spectral properties of optical filters often

call for upgrades of equipment.

The FRET Ratio

Several methods to quantify FRET have been published ( reviewed in (14)). We have

found that methods that rely on extensive mathematical corrections (15, 16) can compound

experimental error in ways that are difficult to rigorously control and derive. Numbers of nearly

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equal value are subtracted and divided, making the methods very sensitive to small errors in the

correction factors.

We prefer a more conventional and straightforward measure of FRET, a FRET ratio, as

an index to the extent of energy transfer (14). The FRET ratio is simply the ratio of the emission

from the acceptor fluorophore divided by the emission from the donor fluorophore while the

sample is excited at the excitation wavelengths of the donor. For the CFP and YFP pair the

FRET ratio is obtained by dividing the fluorescence intensities at 545 nm (YFP emission) by the

fluorescence intensities at 480 nm (CFP emission) while the sample is excited at 440 nm (CFP

excitation) using the excitation and emission filters described above. We will refer to the 545 nm

emission upon 440 excitation as the FRET channel.

The FRET ratio takes advantage of two effects of FRET – increased acceptor emission

and decreased donor emission. First the fluorescence emission from YFP will increase as YFP is

excited by the transfer of excitation energy from CFP. Therefore FRET between CFP and YFP

will increase the emission intensity detected at 545 nm. A FRET interaction also diminishes the

signal from CFP. Without a FRET partner, an excited CFP molecule returns to its ground state

emitting photons with wavelengths which peak around 480 nm. With a FRET partner, the

excited CFP molecule transfers a fraction of that energy to the YFP acceptor, how much

depending on the efficiency of transfer. Thus emission at 480 nm decreases. These two effects

cause the numerator of the FRET ratio to increase and the denominator to decrease, driving the

ratio up.

As described below, the FRET ratio is influenced by several factors in addition to energy

transfer. Some CFP emission tails into the FRET channel. YFP is excited directly, although

inefficiently, at the excitation wavelengths of CFP. Finally changes in the relative amounts of

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CFP and YFP will alter the ratio. Confidence in the FRET ratio as a measure of FRET requires a

careful assessment of the contributions of the spectral overlap and YFP/CFP stoichiometry.

Strain Construction

The first step is the construction of appropriate strains. For FRET analysis the levels of

expression of the CFP and YFP fusions are best controlled by endogenous promoters. This avoids mis-

localization of the fusion, fluctuations in protein levels that often accompany episomal expression, and

the potential to drive artificial protein-protein interactions by mass action under conditions of

overexpression. Several PCR integration systems use homologous recombination to target a

fluorescent protein ORF to the 5' or 3' end of a gene and leave gene expression under the control of the

native promoter. We have mutated plasmids designed by Wach et al. (3) and Prein et al. (2) so that C-

terminal or N-terminal fusions can be made to CFP or YFP. Our CFP is

F64L/S65T/Y66W/N146I/M153T/V163A/N164H relative to GFP (17). Our YFP is

S65G/V68L/Q69K/S72A/T203Y (17). The plasmids to generate the fusions are available from the

YRC. The YRC has made over 50 functional fusions using these systems. We are evaluating other YFP

mutants (11), including the new citrine (18), for improved spectral characteristics over the current YFP

derivative.

Our protocol for making C-terminal fusions is available at our web site:

http://depts.washington.edu/~yeastrc/. Noteworthy is our experience that 40 bp of homology is

sufficient for recombination and insertion of the PCR product into the genome. Also cost savings are

achieved by PAGE purifying only the forward primer containing the homology to the 3’ end of the

ORF. The reverse primer, with homology to the 3’ UTR, is just desalted. Our primers are synthesized

by Integrated DNA Technologies, Inc.

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To make N-terminal fusions, a PCR cassette is targeted by homology to the 5' end of a gene (2).

Both primers are PAGE purified. Integration of this cassette at the 5' end inserts the selectable marker,

G418 resistance, and the GFP variant between the target gene and its promoter. Since integration

temporarily disrupts expression, a diploid strain is transformed and G418 selection isolates hemizygous

cells carrying the cassette. Flanking the G418 resistance marker are a pair of loxP sites. A plasmid with

creA recombinase under control of the GAL1 promoter is then transformed into these cells. Induction of

creA on YP gal plates excises the G418 resistance marker to produce a CFP or YFP ORF fused to the 5'

end of the gene of interest with the sequence of one 32 bp lox site added just upstream of the

fluorescent protein ORF. The strain is then sporulated to yield a haploid strain in which the fusion

protein has now replaced the wild-type protein.

FRET is exceptionally sensitive to the distance between CFP and YFP. In the absence of

detailed knowledge of the structure of the proteins, one cannot predict a priori which fusions will better

position the fluorophores for FRET. Neither can one predict which fusion will be tolerated by the

protein and remain functional. The optimal fusion must be determined empirically for each protein.

Interpreting the FRET ratio requires several careful controls. For a positive control, a protein of

interest fused to a tandem CFP-YFP should give a strong FRET signal. We have designed a plasmid,

pDH18, that contains a tandem YFP and CFP ORF separated by a glycine-alanine linker. This plasmid

is based on the Wach et al. plasmids (3) and has the S. pombe his5 gene as a selectable marker. We

used pDH18 as a PCR template to construct a positive control strain, DHY138, that expresses the

tandem YFP-CFP fused to the histone H4 protein Hhf2p. DHY138 is a heterozygous diploid strain with

the genotype of HHF2::YFP-CFP/HHF2. Fluorescent protein fusions to Hhf2p are bright and easy to

quantify.

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Two negative control strains are needed to characterize the spectral overlap between the

excitation and emission spectra of CFP and YFP. One strain contains a CFP fusion to the donor protein

and the other strain contains a YFP fusion to the acceptor protein. The strains are isogenic except for

the genes encoding the recombinant fusions. These strains serve two purposes. First, since the fusions

are expressed independently, the intensity values in the FRET channel represent a negative control in

which the signal is derived solely from the spectral overlap and the limitations of the optical filter sets.

A second purpose evaluates the relative signal intensities from the CFP and YFP fusions when the

fluorphores are excited at their optimum wavelengths. From these values one assesses the fluctuation in

intensity values from cell to cell for each fusion, and the comparative amount of each fusion. If the

intensity values fluctuate greatly, or if the YFP signal is greater than ~5 fold brighter than the CFP

signal, the evaluation of FRET will be severely compromised. For our general purpose negative

controls we use two heterozygous diploid strains, DHY57 and DHY199, containing HHF2-YFP and

HHF2-CFP, respectively. Both have a wild-type copy of HHF2.

Finally, a set of experimental strains is designed. Each strain carries a donor CFP fusion to one

protein and an acceptor YFP fusion to a protein suspected to closely interact. We tested for contact

between the C-terminal end of Hhf2p and the C-terminal end of an adjacent Hhf2p molecule in the

histone complex by constructing a diploid strain containing Hhf2p-YFP and Hhf2p-CFP in the strain

DHY137 (HHF2-CFP/HHF2-YFP).

Sample Preparation

Imaging of live cells demands that all manipulations are gentle and do not disrupt cell

physiology. We grow cells overnight on YPD plates (19) and in cases where cells carry ade2 and ADE3

we add 0.2 ml of 5 mg/ml adenine to the plates before streaking the strain. The cells have uniformly

low autofluorescence and are growing exponentially as determined by budding index. The cells are

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carefully scraped from the plate and suspended in 300 µL of SD complete medium (19). In this manner

a high density suspension of cells in log phase is obtained without concentrating the cells by

centrifugation.

To make slides, SD complete medium containing 1.2% agarose (SeaKem LE agarose) is melted

and poured into the 0.5 mm depression in concavity slides (PGC Scientifics Corp). The depression

holds roughly 250 µL. A second flat slide is placed over the filled depression and the agar is cooled.

Once cooled, the top slide is slid off leaving a depression filled with a bed of solid SD complete agar at

an appropriate temperature. The concavity slides are cleaned and reused. For more routine work a thin

film 60 nm thick of agarose medium is created on a microscope slide by sandwiching the agarose

between two slides on which Scotch® tape is applied across both ends of one side. Other techniques

include replacing the agarose with 25% gelatin, and including Oxyrase™ in the medium to deplete

oxygen and reduce bleaching (6).

An aliquot (10 µL) of cells is placed onto the agarose bed and a coverslip is pressed down over

the cells. For long observation times the coverslip is sealed at the edges with nail polish to prevent

drying of the agarose. An alternative to nail polish is valap (lanolin:petroleum jelly:paraffin 1:1:1).

The cells lie in one focal plane at the coverslip surface and are embedded in the agar. The agarose pad

supports the cells nutritionally.

As an alternative to live cell imaging, cells can be fixed with formaldehyde or

paraformaldehyde. Fixation results in decreased signals from the fluorescent proteins, but effective

antifade agents can be added to prevent photobleaching. We add formaldehyde to a cell suspension

(prepared as above) to a final concentration of 3.7%. This solution is placed in a 30°C rotator for 15-20

minutes. Formaldehyde fixed cells are adhered to polylysine-coated coverslips, and glycerol with an

antifade agent such as Citifluor™ (Ted Pella Inc.) is used as a mounting medium.

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Optimization of Imaging Parameters

We strongly recommend starting with a positive control and optimizing the imaging technique

to maximize the FRET ratio. Limiting exposure time is crucial for detecting FRET signals. The YFP

acceptor is very sensitive to bleaching by exposure to the 440 nm light used for CFP excitation, with a

half-life of approximately 600 milliseconds in our experiments (Figure 1). Bleaching of CFP is not as

rapid, but still occurs with a half-life of about 3 seconds (Figure 1). Clearly limiting exposure of the

sample to 440 nm light will assist in FRET detection. Even the strong FRET ratio from the Hhf2p-YFP-

CFP tandem fusion is barely detectable after 5 seconds of total exposure (1 s at 500nm, 4 s at 440nm,

Figure 2). We do the following to protect YFP from bleaching:

1) Use transmitted light for focusing.

Light intensity from a transmitted light source (typically a halogen bulb) is negligible compared

to the light intensity generated by a mercury lamp. We have not observed effects on FRET signals from

exposure to transmitted light.

2) Take very short YFP exposures if additional focusing is necessary.

We bin our camera either 2x2 or 3x3 and take 100 millisecond exposures if focusing the

fluorescent signal is necessary. The FRET signal is bleached slowly by YFP excitation light (500 nm)

compared to 440 nm exposure. Therefore the YFP filter set (Ex500 | Em545) is used for additional

fine focusing. The optimal focal plane is found by raising and lowering the stage in small increments

(<400 nm) and locating the position where the pixel values from the YFP emission signal are

maximized.

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3) Take short exposures during data acquisition.

Exposure times are minimized when acquiring data. Many CCD cameras generate 12 bit files

with 4096 gray levels. We do not extend the exposure time to make use of the full range since the

FRET signal is destroyed within 5 seconds of exposure.

4) Take the FRET image before the CFP image.

The order of data acquisition affects the strength of a measured FRET signal. The YFP image

(Ex500|Em545) is taken first, the FRET image (Ex440|Em545) is taken second and the image of CFP

emission (Ex440|Em480) third to avoid bleaching the YFP. Typically only minor bleaching of the CFP

occurs while the FRET image is taken (Figure 1).

Using all of these techniques to maximize the FRET signal, our Hhf2p-YFP-CFP tandem

positive control strain generates a mean FRET ratio of 3.55 with a standard deviation of 0.20 (see

below, “Data Analysis”). This ratio will vary depending on the particular optics and camera used for

detection.

Spectral Overlap

Some fluorescence from CFP reaches into the FRET channel since the broad emission spectrum

of CFP extends beyond 545 nm (14). Thus even without an acceptor YFP present, the FRET ratio has a

value that reflects the amount of fluorescence from CFP that spills over into the FRET channel. In

addition, the excitation spectrum of YFP extends below 440 nm into the excitation wavelengths of CFP

(14). Fluorescence from YFP that comes from direct excitation of YFP also appears in the FRET

channel. The negative control strains identify the contribution of the spectral overlap to the signal in

the FRET channel.

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1. Overlap from CFP

The CFP alone strain is imaged to determine the FRET ratioCFP (Figure 3). The signal intensity

in the FRET channel (Ex440|Ex545) is divided by the signal intensity in the CFP channel

(Ex440|Em480). For the HHF2-CFP/HHF2 diploid strain DHY199, we observe a mean ratio of 1.22

with a standard deviation of 0.072. This ratio will differ from system to system since it depends in part

on the optical filter sets and the quantum efficiency of the CCD camera. Note that CFP does not

contribute any fluorescence to the YFP channel (Figure 3).

2. Overlap from YFP

The amount of fluorescence in the FRET channel for a given amount of YFP (the YFP overlap

factor) is characterized by imaging the YFP-protein fusion strain that does not express the CFP fusion

(Figure 3). Two images are compared: Ex500|Em545 (YFP channel) and Ex440|Em545 (FRET

channel). The YFP overlap factor is the amount of fluorescence in the FRET channel divided by the

amount in the YFP channel. Using our HHF2-YFP/HHF2 diploid strain DHY57, the YFP overlap

factor is 0.26 with a standard deviation of 0.020. This factor is a function of the relative FRET and

YFP exposure times. Therefore the exposure times must remain constant throughout the experiments

for this factor to retain significance. Also note that YFP does not contribute any fluorescence to the

CFP channel (Figure 3).

The Experiment

1. Grow experimental and control strains overnight as described under “Sample Preparation”.

2. Mount cells on slides as described under “Sample Preparation”.

3. Focus with transmitted light as described under “Optimizing Imaging Parameters”.

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4. Take three images of each sample in the following order: a) YFP filter set (Ex500|Em545), b)

FRET filter set (Ex440|Em545), c) CFP filter set (Ex440|Em480). The parameters for image

capture will depend on the strength of the signal above noise and the pattern of intracellular

localization. For the Hhf2p fusions the exposure times were 200 ms for the YFP channel, 400

ms for the FRET channel and 400 ms for the CFP channel. The camera was binned 2x2. The

images obtained for the positive control strain and the experimental strain are shown in

Figure 3.

Data Analysis

Quantification of Signal Intensities

Analysis of the image depends on the pattern of intracellular localization. For the

histones, the total intensity is summed from a region in the center of the histone signal. We

sampled a 700 x 700 nm square (a 5 x 5 pixel square, 2 x 2 camera binning). An example of the

data we acquired is presented in Table 1. The topographical representations of the FRET ratios

(Figures 2 and 4) show a 25 x 25 pixel square centered on the histone signal.

Data Presentation

As a first step in the analysis, the easiest method to display the FRET ratio is to divide the

intensity values in the FRET channel by the intensity values in the CFP channel across the

image. This manipulation is performed by the image analysis software to generate a three-

dimensional topographical display (Figure 4). The view of the display is rotated to emphasize the

value of the FRET ratio at different positions in the image. Different perspectives visually

portray the FRET ratios across the image. In Figure 4 the positive control (HHF2-YFP-

CFP/HHF2) shows the FRET ratios rising from the plane, while the experimental sample

(HHF2-YFP/HHF2-CFP) remains relatively flat. The image reveals significant energy transfer

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in the positive control, but none or very little from the experimental sample. The background

noise was not subtracted because background subtracted values for the CFP channel approach

zero in regions void of CFP and cause the FRET ratio around the edges of the image to fluctuate

greatly.

A quantitative analysis requires extracting the intensity values from a number of images

and calculating a mean FRET ratio. Table 1 shows the data from 8 cells of the positive control

expressing the tandem fusion of Hhf2p-YFP-CFP. For these values a background value,

determined for each cell, has been subtracted. The technique to determine the background values

for an image may vary depending on the nature and distribution of the fluorescent signal. For the

Hhf2p fusions the intensity values from an area of equal size to the sampled histone signal, i.e. a

25 pixel square, were summed. The area was inside the cell but away from the histone signal.

The data for the YFP, FRET and CFP channels were analyzed to yield a mean FRET ratio of

3.55 with a standard deviation of 0.20. The mean YFP/CFP ratio was 1.81 with a standard

deviation of 0.15.

Table 1 also shows the corresponding data for the experimental strain expressing Hhf2p-

YFP and Hhf2p-CFP. The absolute values for the YFP and CFP channels are higher than the

values from the positive control since in the experimental strain there is no wild-type Hhf2p

present to diminish the signal. In the experimental strain the calculated mean FRET ratio was

1.75 with a standard deviation of 0.084. The mean YFP/CFP ratio was 1.39 with a standard

deviation of 0.16.

Data Interpretation

The final step analyzes the data to determine whether the FRET ratio obtained from the

experimental strain reflects energy transfer between CFP and YFP. In the absence of energy transfer, a

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mixture of CFP and YFP proteins will give a signal in the FRET channel. The intensity of that signal

depends in part on the amount of CFP and YFP present. Since that could vary for each donor and

acceptor pair, it must be taken into account for each experiment. The following formula calculates the

ratio expected for a given donor and acceptor if no energy transfer is occurring:

FRET ratioBaseline = FRET ratioCFP + YFP overlap factor x YFP channel

CFP channel

The FRET ratioCFP accounts for the contribution from the CFP fusion protein and is

determined by imaging the strain carrying the CFP fusion alone as described above (“Spectral

Overlap”). The YFP overlap factor accounts for the contribution from the YFP fusion protein and is

determined by imaging the strain carrying the YFP fusion protein alone (see “Spectral Overlap”). The

FRET ratioCFP was 1.22 and the YFP overlap factor was 0.26 for our microscope system and controls.

The ratio of YFP channel/CFP channel is determined from the strains containing both the YFP and CFP

fusion proteins.

Energy transfer is clearly indicated between CFP and YFP in the positive control. Given the

YFP/CFP ratio of 1.81 (Table 1), the calculated FRET ratioBaseline is 1.67 with a standard deviation of

0.011. For the Hhf2p-YFP-CFP labeled histones the observed FRET ratio of 3.55 with a standard

deviation of 0.20 is significantly higher (Table 1). Under ideal in vitro conditions purified CFP-YFP

pairs give a 3 to 4 -fold increase in FRET ratio when the individual proteins are linked by a 25 amino

acid linker (11). The two-fold increase in the observed FRET ratio of the Hhf2p-YFP-CFP over the

calculated FRET ratioBaseline is lower but still easily detectable. The short Gly-Ala linker between the

YFP and CFP in the Hhf2p fusion may impose conformational constraints that limit FRET. However, a

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YFP-CFP tandem Hhf2p fusion with a 4 x (Gly-Ala) linker between the YFP and CFP did not

significantly change the FRET ratio.

Based on the crystal structure of the nucleosome from Xenopus (20) energy transfer is predicted

between Hhf2p-CFP and Hhf2p-YFP in the experimental strain. Histones 1 thru 4 are assembled into an

octamer that forms the core of the nucleosome. Each octamer contains a pair of each of the four

histones. The C-terminal ends of the two histone H4 proteins are 30 Å apart in the solved structure.

This distance would allow energy transfer between CFP and YFP.

The mean observed FRET ratio for the Hhf2p-YFP/Hhf2p-CFP complex was 1.75 with a

standard deviation of 0.084 (Table 1). The calculated FRET ratioBaseline was 1.58 with a standard

deviation of 0.11. The 10% increase in the observed FRET ratio suggests energy transfer between CFP

and YFP. The increase is less than might be predicted from the proximity of the two histone H4

molecules in the crystal structure, but as described below several factors could contribute to a lower

than expected value. For comparison, FRET ratio increases of 4%, 10%, and 250% were used to predict

protein-protein interactions in other published work (16, 21, 22).

Several factors can decrease the FRET signal for proteins that interact. First, although the

proteins may be close, the orientation of the CFP and YFP may not be optimal for FRET. Second,

when examining the pairing of a protein with itself, random mixing will dilute the signal by half since a

tagged protein cannot FRET with a protein tagged similarly. In the case of Hhf2p, the experimental

strain is a diploid in which one copy of HHF2 is tagged with CFP and one copy is tagged with YFP.

Thus, statistically the maximum theoretical FRET signal will be reduced by half simply by the random

process of selection of histones into the nucleosome. Finally the FRET ratio can be depressed if

untagged proteins can substitute for the tagged proteins and dilute the amount of tagged proteins in the

complex. In the case of Hhf2p the FRET signal will be reduced by the presence of Hhf1p. Hhf1p is a

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histone H4 protein that is identical to Hhf2p. HHF1 expression is 5 to 7 fold less than HHF2 (23), so

the reduction in FRET should be minor. These points indicate that a lack of FRET between proteins

does not argue against interaction; however, the observation of FRET between two tagged proteins

demonstrates their close proximity.

Conclusion

Live cell FRET detection in yeast is in its infancy, and there are significant complications

with the CFP and YFP pair. Spectral overlap between the excitation and emission spectra

complicates analysis. The extreme sensitivity of the current YFP to bleaching constrains image

acquisition. Detection of a FRET signal is limited by background cellular autofluorescence and

the relatively weak fluorescent signal intensities. Dynamic intracellular conditions, such as

changes in pH or protein concentrations, can complicate the interpretation of experimental data.

However, with careful controls FRET is a powerful indication of protein-protein interaction. In

instances where interactions give robust FRET signals, FRET is a valuable tool to study the

dynamic spatial and temporal behavior of protein-protein interactions in living cells. Future

improvements in the spectral properties of CFP and YFP will increase the general applicability

of FRET to study a broad range of protein-protein interactions in yeast.

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Table 1. FRET ratios for the positive control strain and the experimental straina

Positive Control: Hhf2p-YFP-CFP / Hhf2p:

Experimental: Hhf2p-YFP / Hhf2p-CFP:

a Images were captured as described in Figure 3.

Cell YFP channel FRET channel CFP channel YFP/CFP FRET Ratio1 33649 42030 23355 1.44 1.802 28993 38653 22821 1.27 1.693 30814 34492 17648 1.75 1.954 29355 37999 21875 1.34 1.745 22720 27367 15853 1.43 1.736 22661 29201 17293 1.31 1.697 19662 27871 15990 1.23 1.748 18493 23409 14043 1.32 1.67

Mean 1.39 1.75St.D. 0.16 0.084

Cell YFP channel FRET channel CFP channel YFP/CFP FRET Ratio1 20781 40326 11615 1.79 3.472 15174 32506 9791 1.55 3.323 12723 27948 7230 1.76 3.874 13965 26629 7303 1.91 3.655 16752 33622 9531 1.76 3.536 12172 23268 6842 1.78 3.407 23523 42753 11183 2.10 3.828 16247 29272 8821 1.84 3.32

Mean 1.81 3.55St.D. 0.15 0.20

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Figure Legends

Figure 1: CFP and YFP bleaching by 440 nm light. The two strains examined were DHY57 (HHF2-

YFP/HHF2) and DHY199 (HHF2-CFP/HHF2). The strains were grown and imaged as described in the

text. Each curve summarizes data collected from 4 different cells. Data was sampled by summing total

intensity inside a 5x5 pixel square. Background was determined from a 5x5 pixel square inside each

cell but away from the histone signal. l , CFP bleaching at 440 nm, repeated 400 ms CFP exposures

were captured. n , YFP bleaching at 440 nm. To mimic the conditions during a FRET experiment, we

repeatedly exposed the sample to a 200 ms YFP exposure followed by an 800 ms CFP exposure.

Bleaching of the YFP during the YFP exposures is negligible. The error bars show the standard

deviation.

Figure 2: Rapid bleaching of the FRET signal during sequential image capture. Strain DHY138

(HHF2::YFP-CFP/HHF2) was grown and imaged as described in the text. Six sequential time points

were taken. At each time point a 200 ms YFP channel, 400 ms FRET channel and 400 ms CFP channel

exposure were captured. Each FRET channel image was divided by the CFP channel image to generate

six sequential FRET ratio images. Pixel values in the ratio images are the FRET ratios at each position

in the image. A 25x25 pixel square centered on the histone signal was sampled. Data from the first,

third and fifth ratio images are shown. The numerical range of the FRET ratios in the sampled region is

indicated by the grayscale. Elapsed total exposure time is indicated beside the plots.

Figure 3: Images of our negative and positive control strains compared with the experimental strain.

Strains DHY57 (YFP overlap control), DHY199 (CFP overlap control) and DHY138 (positive control)

were compared to DHY137 (HHF2-CFP/HHF2-YFP) (brief strain descriptions in text). Samples were

prepared for microscopy as described in text. Three images of each strain were captured in this order: a

200 ms YFP channel exposure, a 400 ms FRET channel exposure and a 400 ms CFP channel exposure.

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Spectral overlap from both CFP and YFP is observed in the FRET channel. Overlap from CFP into the

YFP channel and overlap from YFP into the CFP channel is not observed. The DHY57 images are

scaled from 100-2500. The DHY199 images are scaled from 100-900. The images from DHY137 and

DHY138 are scaled 100-2000. Both the YFP and CFP fusion proteins are functional and the fluorescent

protein fusions to Hhf2p do not obviously affect the localization of Hhf2p.

Figure 4: Graphical displays of FRET ratios in sampled regions. Images of DHY137 and

DHY138 were captured as described in Figure 3. Ratio images were generated as described in

Figure 2. A 25x25 pixel square centered on the histone signals in the FRET ratio images was

sampled. The left panels show data in the same orientation as the raw images. Squares

representing pixels are shaded gray to indicate the FRET ratio at each position. The images on

the right are rotations of the left panels.

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26

Figure 2

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Figure 3

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Figure 4