Paul Gavin# and Mark Prescott#, Ph.D
Daren J. Fyfe, Ph.D*
# Department of Biochemistry and Molecular Biology, Monash University, Clayton campus Victoria 3800, Australia
* Technical assistance: Varian Australia Pty Ltd, Mulgrave, Victoria 3170, Australia E-mail: firstname.lastname@example.org
Fluorescence resonance energy transfer (FRET) is a non-destructive, spectroscopic approach that can be used to monitor the proximity and angular orientation of donor and acceptor fluorophores in living cells1. The resonant energy of an excited donor fluorophore (in this example blue fluorescent proteinBFP) is absorbed by an acceptor fluorophore (green fluorescent proteinGFP) providing that donor and acceptor are in close proximity (between 1080 ngstroms2) (Figure 1). Emission spectra of donor fluorophores must significantly overlap the absorption spectra of the acceptor, while overlap between respective absorption and emission spectra of donor and acceptor should be minimized 3 .
Due to the attractive attributes of GFP (previously described in fluorescence application note No. 5)4, the application of FRET to GFP and GFP variants has become a powerful tool to monitor interactions at the protein level, within an intact cell or organism. It is possible to use FRET to measure conformational changes in a molecule tagged with two GFP variants in response to the binding of ligands such as calcium1, or the interaction between separate proteins, each tagged with a specific GFP5. Studies such as these present the unique opportunity to study subtle relationships and dynamic interactions between proteinsin living cells.
The present study aimed to detect and monitor changes in FRET between BFP and GFP in cytosolic lysates of yeast cells using the Varian Cary Eclipse.
Materials and Methods
(For part numbers see reference6)
Varian Cary Eclipse fluorescence spectrophotometer
Peltier-thermostatted multicell holder (with electromagnetic stirring)
Magnetic stirrer bars
YRD15 (MATa, his3, ura3, leu2, p+) of the yeast S. cerevisiae was the parental strain used in this study. A gene cassette was constructed encoding BFP and GFP linked by a 27 amino acid peptide linker that contains a recognition site for the protease trypsin. This cassette was cloned into the yeast expression plasmid pAS1N for cytosolic expression and transformed into the yeast strain YRD15 as previously described7. Transformants were plated out on yeast minimal medium (0.75% yeast minimal medium w/o amino acids, 2% glucose, 1.5% agar) with growth supplements as required and grown at 28C for 35 days.
Yeast cells were washed twice in 1ml MilliQ water before being lysed using Y-PER (Progen) as per the manufacturers instructions. Lysates were preferred over whole cells to allow protease digestion of the peptide linker. Y-PER lysates (10l) were diluted with 1.2ml Tris/HCl pH 8 and placed in disposable fluorescence cuvettes (Sarstedt) of the multicell holder positioned within the sample chamber of the Varian Cary Eclipse. The temperature within the cuvettes was set to 25C to promote cleavage of the peptide linker by trypsin (Figure 2). Using the Scan application, BFP was specifically excited using light of 360nm, and emission spectra for the fusion protein were recorded from the range 400550nm. Further emission scans were recorded over time after the addition of 0.25g trypsin.
Emission spectra of the BFP-GFP fusion protein following 360nm excitation are shown in Figure 3. An initial spectrum was taken at time = 0 min, then trypsin was added and spectra were recorded at the times indicated. GFP emission (~510nm) is seen upon specific excitation of BFP alone (360nm), indicative of FRET. Spectral characteristics of FRET (indicated by the green peak at 510nm) progressively disappeared following the addition of trypsin, which cleaves the peptide linker that tethers the GFPs. A small increase is seen in BFP emission as FRET diminishes.
An ideal strategy to monitor the interactions of proteins in living systems involves detection of FRET between BFP and GFP bound to target species of interest. For optimum selectivity and sensitivity of detection of fluorescence in applications such as this it is necessary to minimise (a) detection of cellular autofluorescence and (b) photobleaching. These issues are addressed by internal filters (on both excitation and emission monochromators) and the Cary Eclipse xenon flash lamp respectively. The issue of photobleaching is extremely important and is discussed in a separate application note.
The data indicate (Figure 3) that FRET could be accurately monitored in cytosolic lysates from yeast cells. Cleavage of the BFP-GFP fusion using trypsin (Peltier temperature controlled at 25C) demonstrated that GFP emission was due to FRET and not to direct stimulation of GFP by the excitation wavelength. This is demonstrated by the scans shown in Figure 3 that depict the green (FRET) peak becoming smaller (as BFP is cleaved from GFP) with successive scans over a period of 33 minutes.
Conclusion The Varian Cary Eclipse with Peltier temperature control and multicell accessories provide a simple and accurate assembly with which to monitor cell function at the protein level in cytosolic lysates from yeast cells. The opportunity now exists to use this model as a platform with which to investigate protein-protein interactions in response to external or internal stimuli in living cells.