Sarah F. Martin, School of Physics and Astronomy, University of St. Andrews, North Haugh, St. Andrews, KY16 9SS, UK; Michael H. Tatham, College of Life Science, University of Dundee, Dow Street, Dundee, DD1 5EH, UK, 1/2008
Protein interactions regulate essential cellular functions, ranging from subcellular transport and cell structure formation to DNA transcription and translation. Failure of the sophisticated cross-talk within complex protein cycles can lead to diseases such as Alzheimers, diabetes and certain forms of cancer.
The study of these interactions has been significantly advanced by the discovery of the genetic structure of the green fluorescent protein (GFP) in the jellyfish Aequorea Victoria(1), enabling fluorescent labelling. Furthermore, the introduction of mutations into GFP has allowed the generation of fluorescent probes with altered spectral properties (e.g. cyan FP and yellow FP), that facilitate the use of fluorescence resonance energy transfer (FRET) to indicate the proximity of tagged molecules.
Energy transfer is observed as a decrease in emission intensity and fluorescence lifetime of a higher energy (bluer “donor”), and an increase in emission and lifetime of a lower energy (redder “acceptor”) fluorophore. For this, the emission spectrum of the donor probe must overlap considerably the absorption spectrum of the acceptor probe.
When fluorescently tagged proteins interact, the distance between the attached fluorescent probes is effectively reduced. This leads to energy transfer and related changes in the combined fluorescence spectra.
In this application note, we study the small ubiquitin-like modifier SUMO1 and SUMO2 and their processing by the protease SenP1. We developed an in vitro FRET-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-specific C-terminal hydrolase activity. The measurements were performed on BMG LABTECH´s NOVOstar (Figure 1)
Fig. 1: NOVOstar from BMG LABTECH
cDNA cloning and protein expression and purification
A plasmid (pHis-TEV-30a-YFP-ECFP) that contains cDNA for the two fluorophores yellow fluorescent protein (YFP, a kind gift from A. Miyawaki, RIKWN, Japan) and enhanced cyan-fluorescent protein (ECFP) was constructed. cDNA for SUMO1 (NCBI Entrez Protein CAA67898) and SUMO2 (NCBI Entrez Protein CAG46970) were inserted into the plasmid(2).
Proteins 6His-TEV-YFP-SUMO1-ECFP and 6His-TEV-YFP-SUMO2-ECFP were expressed in Escherichia coli B834de3 grown in L-broth to an optical density at 600 nm of 0.6-0.8 by induction with 1 mM isopropyl ß-D-thiogalactoside for 16 h at 25°C. Soluble 6His-TEV-SUMO-FL-ECFP proteins were purified to ~90% homogeneity2 also confirmed by mass spectrometry.
FRET assay data acquisition and analysis
FRET-based SUMO processing assays were conducted by measuring the emission intensity of ECFP at 480 nm (480-10 filter) and of YFP at 530 nm (530-10 filter) with an excitation wavelength of 405 nm (405-20 filter) on the NOVOstar. 20 µL of fluorescent SUMO protein was added to 5-10 µL of protease-containing solution leading to 25-30 µL final assay volume, buffered in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM ß-mercaptoethanol, 0.1 mg/mL bovine serum albumin. After mixing, initial rate data were measured in short intervals. After that all reactions were read at 3- to 8-min intervals until completion.
As YFP-SUMO-ECFP is processed by the protease, the FRET signal will decrease and the direct ECFP fluorescence will increase due to reduced energy transfer to YFP (Figure 2). The ratio of 530nm/480nm can be used as an internally controlled and reliable measurement of the absolute quantities of SUMO substrate in the assays if the “uncleaved” and “100% cleaved” 480 nm and 530 nm measurements are known.
Fig. 2: Principle of the FRET - based SUMO assay, FRET takes place from ECFP (Donor, excitation light of 405 nm) to YFP (Acceptor, emission measured at 530 nm). Once cleaved by SUMO protease, the distance between the fluorophores is increased beyond a FRET-sensitive distance and thus ECFP emission measured at 480 nm is increased while the YFP FRET emission is reduced.
A valuable application of a FRET based SUMO processing assay would be its ability to provide quantitative kinetic information on the progress of SUMO processing reactions. To investigate these possibilities, assays containing YFP-SUMO1-ECFP were monitored over time after addition of varying concentration of the SUMO protease SenP1 (Fig.3).
Fig. 3: FRET-based time-course analysis of SUMO1 cleavage by SenP1 in the presence of different SenP1 concentrations. For each reaction the emission at 480 nm and 530 nm was measured every 1.5 s for the first minute and then every 6 min. The ratio of 480/530 nm was subsequently converted to nanomols of substrate cleaved.
The observed rates of SUMO processing were found to be proportional to both the duration of incubation and the concentration of protease, which is consistent with enzyme catalysed proteolysis dynamics. Repetition of this analysis using YFP-SUMO2-ECFP also shows a dependence upon SenP1 concentration and time (data not shown).
Fig. 4: Comparison of the initial rates of SUMO1 and SUMO2 processing with respect to SenP1 concentration
Both figures also show that there are significant differences between SUMO-1 and SUMO-2 processing by SenP1, with SUMO-2 being a significantly poorer substrate than SUMO-1. This is consistent with published observation using the wild-type proteins(3) that show that SenP1 has a preference for processing SUMO-1 over SUMO-2 and thus confirms the assay to be biologically relevant.
The FRET SUMO processing assay can be enabled for high-throughput screening of potential SUMO protease inhibitors. Fig. 5 shows the effect of 50 mM EDTA, 10 mM or 50 mM iodoacetamide on SenP1 activity.
Fig.5: Progress curve of FRET SUMO protease assays containing 20 µL of 1 µM YFP-SUMO1-ECFP cleaved by 0.625 nM SenP1 in the presence of EDTA and varying concentrations of iodoacetamide (IA)
As expected, because SenP1 is a cysteine protease with no dependence on metal ions, EDTA has no effect on catalysis, while there is a dose-dependent effect of the iodoacetamide which carboxymethylates the active site cysteine). This simple test demonstrate the potential application of the technique to screening for protease inhibitors.
The FRET-based SUMO processing assay described here significantly improves upon existing methods by giving paralog-specific, quantitative and kinetic information on SUMO processing reactions.
Furthermore, the fluorescent substrates are easily expressed and purified from bacteria with a relatively high yield. The described assay has applications in SUMO protease characterization, enzyme kinetic analysis and high-throughput inhibitor screening.
The NOVOstar is the ideal platform to perform this FRET-based assay, because it has a unique pipetting system that can add very small amounts of substance (<1 µL) followed by instantaneous reading.
The multifunctional microplate reader NOVOstar is able to measure fluorescence intensity, luminescence, absorbance, and fluorescence polarization and is also equipped with incubation, shaking and user-defined kinetic sampling.