Direct observation of resonance tryptophan-to-chromophore energy transfer in visible fluorescent proteins

Visible fluorescent proteins from Aequorea victoria contain next to the fluorophoric group a single tryptophan residue. Both molecules form a single donor-acceptor pair for resonance energy transfer (RET) within the protein. Time-resolved fluorescence experiments using tryptophan excitation have shown that RET is manifested by a distinct growing in of acceptor fluorescence at a rate characteristic for this process. In addition, time-resolved fluorescence anisotropy measurements under the same excitation-emission conditions showed a correlation time that is similar to the time constant of the same RET process with the additional benefit of gaining information on the relative orientation of the corresponding transition dipoles.     

Development of a baculovirus-based fluorescence resonance energy transfer assay for measuring protein-protein interaction.

A new baculovirus-based fluorescence resonance energy transfer (Bv-FRET) assay for measuring multimerization of cell surface molecules in living cells is described. It has been demonstrated that gonadotropin-releasing hormone receptor (GnRH-R) was capable of forming oligomeric complexes in the plasma membrane under normal physiological conditions. The mouse gonadotropin-releasing hormone receptor GnRH-R was used to evaluate the efficiency and potential applications of this assay. Two chimeric constructs of GnRH-R were made, one with green fluorescent protein as a donor fluorophore and the other with enhanced yellow fluorescent protein as an acceptor fluorophore. These chimeric constructs were coexpressed in an insect cell line (BTI Tn5 B1-4) using recombinant baculoviruses. Energy transfer occurred from the excited donor to the acceptor when they were in close proximity. The association of GnRH-R was demonstrated through FRET and the fluorescence observed using a Leica TSC-SPII confocal microscope. FRET was enhanced by the addition of a GnRH agonist but not by an antagonist. The Bv-FRET assay constitutes a highly efficient, reliable and convenient method for measuring protein-protein interaction as the baculovirus expression system is superior to other transfection-based methods. Additionally, the same insect cell line can be used routinely for expressing any recombinant proteins of interest, allowing various combinations of molecules to be tested in a rapid fashion for protein-protein interactions. The assay is a valuable tool not only for the screening of new molecules that interact with known bait molecules, but also for confirming interactions between other known molecules.     

Imaging fluorescence resonance energy transfer between two green fluorescent proteins in living yeast

We show that fluorescence resonance energy transfer between two mutants of the green fluorescent protein (GFP) can be monitored by imaging microscopy in living yeast. This work is based on the constitutive expression of a GFP-containing fusion protein and the inducible expression of the tobacco etch virus (TEV) protease. In the fusion protein, the P4.3 GFP mutant is linked to the YS65T GFP mutant by a spacer bearing the TEV protease-specific cleavage site.     

Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins.

BACKGROUND: The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. METHODS: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. RESULTS: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. CONCLUSIONS: We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.     

Concatenation of cyan and yellow fluorescent proteins for efficient resonance energy transfer

Highly efficient fluorescence resonance energy transfer between cyan(CFP) and yellow fluorescent proteins (YFP), the cyan- and yellow-emitting variants of the Aequorea green fluorescent protein, respectively, was achieved by tightly concatenating the two proteins. After the C-terminus of CFP and the N-terminus of YFP were truncated by 11 and 5 amino acids, respectively, the proteins were fused through a leucine-glutamate dipeptide. The resulting chimeric protein, which we called Cy11.5, exhibited a simple emission spectrum that peaked at 527 nm when the protein was excited at 436 nm. The time-resolved emission of Cy11.5 was measured using a streak camera. After excitation of Cy11.5 with a 400 nm ultrashort pulse, a fast decay of the CFP emission and a concomitant rise of the YFP emission were observed with a lifetime of 66 ps. By contrast, the emission from CFP alone showed a decay component with a lifetime of 2.9 ns. We concluded that in fully folded Cy11.5 molecules, intramolecular FRET occurred with an efficiency of 98%. Importantly, most Cy11.5 molecules were properly folded, and the protein was highly resistant to all of the tested proteases. In living cells, therefore, Cy11.5 behaved as a single fluorescent protein with a broad excitation spectrum. Moreover, Cy11.5 was used as an optical highlighter after photobleaching of YFP. When HeLa cells expressing Cy11.5 were irradiated at 514.5 nm, a 10-fold increase in the 475 nm fluorescence intensity was observed. These features make Cy11.5 useful as an optical highlighter and a new-colored fluorescent protein for multicolor imaging.     

Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions

A substantial range of protein-protein interactions can be readily monitored in real time using bioluminescence resonance energy transfer (BRET). The procedure involves heterologous coexpression of fusion proteins, which link proteins of interest to a bioluminescent donor enzyme or acceptor fluorophore. Energy transfer between these proteins is then detected. This protocol encompasses BRET1, BRET2 and the recently described eBRET, including selection of the donor, acceptor and substrate combination, fusion construct generation and validation, cell culture, fluorescence and luminescence detection, BRET detection and data analysis. The protocol is particularly suited to studying protein-protein interactions in live cells (adherent or in suspension), but cell extracts and purified proteins can also be used. Furthermore, although the procedure is illustrated with references to mammalian cell culture conditions, this protocol can be readily used for bacterial or plant studies. Once fusion proteins are generated and validated, the procedure typically takes 48-72 h depending on cell culture requirements.     

Direct detection of phospholamban and sarcoplasmic reticulum Ca-ATPase interaction in membranes using fluorescence resonance energy transfer

We used fluorescence resonance energy transfer (FRET) to detect and quantitate the interaction of the sarcoplasmic reticulum Ca-ATPase (SERCA) with phospholamban (PLB) in membranes. PLB inhibits SERCA only at submicromolar Ca. It has been proposed that relief of inhibition at micromolar Ca is due to dissociation of the inhibitory complex. To test this hypothesis, we co-reconstituted donor-labeled SERCA and acceptor-labeled I40A-PLB (superinhibitory, monomeric PLB mutant) in membranes of defined lipid and protein composition, with full retention of Ca-dependent ATPase activity and inhibitory regulation by PLB. FRET from SERCA to PLB was measured as a function of membrane concentrations of PLB and SERCA, and functional activity was measured on the same samples. The data revealed clearly that the stoichiometry of binding is one PLB per SERCA, and that binding is a strict function of the ratio of total PLB to SERCA in the membrane. We conclude that the dissociation constant of PLB binding to SERCA is far less than physiological PLB membrane concentrations. Binding at low Ca (pCa 6.5), where I40A-PLB inhibits SERCA, was virtually identical to that at high Ca (pCa 5.0), where no inhibition was observed. However, the limiting energy transfer at saturating PLB was less at high Ca, indicating a greater donor-acceptor distance. We conclude that (a) the affinity of PLB for SERCA is so great that PLB is essentially a SERCA subunit under physiological conditions and (b) relief of inhibition at micromolar Ca is due to a structural rearrangement within the SERCA-PLB complex, rather than dissociation.     

Cell surface detection of membrane protein interaction with homogeneous time-resolved fluorescence resonance energy transfer technology

Direct or indirect interactions between membrane proteins at the cell surface play a central role in numerous cell processes, including possible synergistic effects between different types of receptors. Here we describe a method and tools to analyze membrane protein-protein interaction at the surface of living cells. This technology is based on the use of specific antibodies directed against each partner and labeled either with europium cryptate or with Alexa Fluor 647. This allows the measurement of a fluorescence resonance energy transfer (FRET) signal in a time-resolved manner if both antibodies are in close proximity. This approach is here validated using the heterodimeric gamma-aminobutyrate B receptor as a model. We show that after washing out the unbound antibodies, the time-resolved FRET signal can be measured together with the expression level of both partners via the quantification of the donor and the acceptor fluorophores bound to the cells. Thanks to the high sensitivity of this method and to the low concentration of antibodies required, we show that the signal can also be measured directly after the incubation period without washing out the unbound antibody (homogeneous time-resolved FRET). As such, this method is highly sensitive, reproducible, and compatible with the development of high-throughput screening protocols.     

Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases

Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these potential functions, the authors designed and used a protease substrate fluorescence resonance energy transfer (FRET) library comprising 115 short d- and l-amino-acid-containing fluorogenic substrates as a tool to generate proteolytic profiles for a wide range of bacteria. Bacterial specificity of the d-amino acid substrates was confirmed using enzymes isolated from both eukaryotic and prokaryotic organisms. Interestingly, bacterial proteases that are known to be involved in housekeeping and nutrition, but not in virulence, were able to degrade substrates in which a d-amino acid was present. Using our FRET peptide library and culture supernatants from a total of 60 different bacterial species revealed novel, bacteria-specific, proteolytic profiles, although in-species variation was observed for Pseudomonas aeruginosa, Porphyromonas gingivalis, and Staphylococcus aureus. Overall, the specific characteristic of our substrate peptide library makes it a rapid tool to high-throughput screen for novel substrates to detect bacterial proteolytic activity.     

Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET)

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set.     

Use of fluorescence resonance energy transfer for rapid detection of enteroviral infection in vivo

Enteroviruses can be easily transmitted through the fecal-oral route and cause a diverse array of clinical manifestations. Recent outbreaks associated with enteroviral contamination in aquatic environments have called for the development of a more efficient and accurate virus monitoring system. To develop a simple, rapid, and direct method for identifying enteroviral infections, we generated a fluorescent reporter system in which genetically engineered cells express a hybrid fluorescent indicator composed of a linker peptide, which is exclusively cleaved by the 2A protease (2A(pro)), flanked with a cyan fluorescent protein (CFP) and a yellow fluorescent protein undergoing fluorescence resonance energy transfer. The covalent linkage between two fluorophores is disrupted due to 2A(pro) activity upon viral infection, which results in an increase in CFP intensity. This allows the rapid (within 7.5 h) detection of very low numbers (10 PFU or fewer) of infectious enteroviruses.     

In vitro observation of the molecular interaction between NodD and its inducer naringenin as monitored by fluorescence resonance energy transfer

At initial stages in the Rhizobium legume symbiosis, most nodulation genes are controlled by NodD protein and plant inducers. Some genetic studies and other reports have suggested that NodD may be activated by its direct interaction with plant inducers. However, there has been no molecular evidence of such an inducing interaction. In this paper, we used fluorescence resonance energy transfer technique to see whether such an interaction exists between NodD and its activator, naringenin, in vitro. The tetracysteine motif (Cys-Cys-Pro-Gly-Cys-Cys) was genetically inserted into NodD to label NodD with 4',5'-bis(1,3,2-dithioarsolan-2-yl) fluorescein (FlAsH). Naringenin was labeled with fluorescein by chemical linking. In the fluorescence resonance energy transfer experiments in vitro, the fluorescence intensity of one acceptor, NodD(90R6)-FlAsH, increased by 13%. This suggests that NodD may directly interact with inducer naringenin in vitro and that the reaction centre is likely near hinge region 1 of NodD.     

A vinblastine fluorescent probe for pregnane X receptor in a time-resolved fluorescence resonance energy transfer assay

The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows the binding of many drugs and drug leads to it, possibly causing undesired drug–drug interactions. Therefore, it is crucial to evaluate whether lead compounds bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. One fluorescent PXR probe is currently commercially available; however, because its chemical structure is not publicly disclosed, it is not optimal for studying ligand–PXR interactions. Here we report the characterization of BODIPY FL–vinblastine, generated by labeling vinblastine with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), as a high-affinity ligand for human PXR with a K_d value of 673 nM. We provide evidence that BODIPY FL–vinblastine is a unique chemical entity different from either vinblastine or the fluorophore BODIPY FL in its function as a high-affinity human PXR ligand. We describe a BODIPY FL–vinblastine-based human PXR time-resolved fluorescence resonance energy transfer assay, which was used to successfully test a panel of human PXR ligands. The BODIPY FL–vinblastine-based biochemical assay is suitable for high-throughput screening to evaluate whether lead compounds bind to PXR.     

Förster distances for fluorescence resonant energy transfer between mCherry and other visible fluorescent proteins

We present, for the red fluorescent protein mCherry acting as both fluorescence resonant energy transfer (FRET) donor and acceptor, Förster critical distance (r₀) values with five important visible fluorescent protein (VFP) variants as well as with itself. The pair EYFP-mCherry exhibits an r₀ of 5.66 nm, equaling or exceeding any combination of VFPs reported previously. Moreover, mCherry should be an excellent chromophore for homo-FRET with an r₀ of 5.10 nm for energy transfer between two mCherry moieties. Finally, mCherry exhibits higher r₀ values than does DsRed. These characteristics, combined with mCherry’s rapid folding and excellent spectral properties, suggest that mCherry constitutes a valuable long-wavelength hetero-FRET acceptor and probe for homo-FRET experiments.     

In vivo interaction between dynamitin and MacMARCKS detected by the fluorescent resonance energy transfer method

Dynamitin is a subunit of the dynactin complex regulating microtubule-dependent motor functions, and MacMARCKS (Macrophage-enriched myristoylated alanine-rich protein kinase C substrate) is a major protein kinase C substrate regulating integrin activation. The interaction between dynamitin and MacMARCKS has been implicated in integrin-dependent cell spreading. However, the in vivo interaction of these two proteins in living cells has not been demonstrated. Spatial and temporal information about the interaction is also lacking. In this study, we used the fluorescent resonance energy transfer method to demonstrate in vivo interaction between MacMARCKS and dynamitin with cyan fluorescent protein (CFP)-conjugated dynamitin as the donor fluorophore and yellow fluorescent protein (YFP)-conjugated MacMARCKS as the acceptor fluorophore. The interaction of these two fusion proteins was studied both in vitro and in vivo, and typical fluorescent resonance energy transfer was observed; the CFP emission peak increased while the YFP emission peak decreased when protein interaction was abolished. Spatial and temporal information was obtained in RAW macrophage cells. In resting macrophage cells, dynamitin-MacMARCKS interaction is concentrated at the cell periphery, although the majority of dynamitin is distributed at the perinuclear region of the cells. When cells were treated with phorbol 12-myristate 13-acetate, both proteins concentrated to perinuclear regions of the cells, and yet the interaction disappeared as the cell spread. Similar events were also observed in 293 cells. Thus, we conclude that dynamitin and MacMARCKS indeed interact in living cells.     

Detection of the swings of the lever arm of a myosin motor by fluorescence resonance energy transfer of green and blue fluorescent proteins

The "lever-arm" model of a myosin motor predicts that the lever-arm domain in the myosin head tilts and swings against the catalytic domain during ATP hydrolysis, resulting in force generation. To investigate if this "swing" of the lever arm really occurs during the hydrolysis of ATP, we employed fluorescence resonance energy transfer (FRET) between two fluorescent proteins [green (GFP) and blue (BFP)] fused to the N and C termini of the Dictyostelium myosin-motor domain. FRET measurements showed that the C-terminal BFP in the fusion protein first swings against the N-terminal GFP at the isomerization step of the ATP hydrolysis cycle and then swings back at the phosphate-release step. Because the C-terminal BFP mimics the motion of the lever arm, the result indicates that the lever arm swings at the specific steps of the ATP hydrolysis cycle, i.e., at the isomerization and phosphate-release steps. The latter swing may correspond to the power stroke of myosin, while the former may be related to the recovery stroke.     

Amplified detection of cocaine based on strand-displacement polymerization and fluorescence resonance energy transfer

Cocaine is one of the most abused drugs in the United States and is potentially dangerous when consumed in excess. Its detection is thus important in many areas in the fight against drug trafficking. We have developed an amplified detection method for cocaine based on a strand-displacement polymerization reaction using aptamer recognition. The system mainly consists of a hairpin probe with Cy5 labeled on its 3' end, a primer with FAM labeled on its 5' end, and polymerase. The aptamer sequence is integrated into the 5'-section of the hairpin probe. The primer is designed complementary to the 3' end of the hairpin probe, which is also part of the hairpin stem region. The cocaine induced reaction cycle generates product for detection and thus for signal amplification. The detection limit of this method is 200 nM in about 16 min and the specificity of this approach is excellent. We believe that this strategy will be useful for the development of analytical schemes for a variety of aptamers for small molecules, metal ions, and proteins. This simple scheme employing the strand-displacement polymerization reaction may find wide application in forensic analysis, environmental monitoring, and clinical diagnostics.     

Dynamics of the UvrABC nucleotide excision repair proteins analyzed by fluorescence resonance energy transfer

UvrB plays a key role in bacterial nucleotide excision repair. It is the ultimate damage-binding protein that interacts with both UvrA and UvrC. The oligomeric state of UvrB and the UvrAB complex have been subject of debate for a long time. Using fluorescence resonance energy transfer (FRET) between GFP and YFP fused to the C-terminal end of Escherichia coli UvrB, we unambiguously show that in solution two UvrB subunits bind to UvrA, most likely as part of a UvrA2B2 complex. This complex is most stable when both UvrA and UvrB are in the ATP-bound form. Analysis of a truncated form of UvrB shows that binding to UvrA promotes dimerization of the two C-terminal domain 4 regions of UvrB. The presence of undamaged DNA leads to dissociation of the UvrA2B2 complex, but when the ATPase site of UvrB is inactivated, the complex is trapped on the DNA. When the complex is bound to a damaged site, FRET between the two UvrB subunits could still be detected, but only as long as UvrA remains associated. Dissociation of UvrA from the damage-bound UvrB dimer leads to the reduction of the magnitude of the FRET signal, indicating that the domain 4 regions no longer interact. We propose that the UvrA-induced dimerization of the domain 4 regions serves to shield these domains from premature UvrC binding. Only after specific binding of the UvrB dimer to a damaged site and subsequent release of UvrA is the contact between the domain 4 regions broken, allowing recruitment of UvrC and subsequent incisions.     

Cyclic AMP-dependent protein kinase binding to A-kinase anchoring proteins in living cells by fluorescence resonance energy transfer of green fluorescent protein fusion proteins.

A-kinase anchoring proteins tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The purpose of this study was to use fluorescence resonance energy transfer to monitor binding events in living cells between the type II regulatory subunit of PKA (RII) and the RII-binding domain of the human thyroid RII anchoring protein (Ht31), a peptide containing the PKA-binding domain of an A-kinase anchoring protein. RII was linked to enhanced yellow fluorescent protein (EYFP), Ht31 was linked to enhanced cyan fluorescent protein (ECFP), and these constructs were coexpressed in Chinese hamster ovary cells. Upon excitation of the donor fluorophore, Ht31.ECFP, an increase in emission of the acceptor fluorophore, RII.EYFP, and a decrease in emission from Ht31.ECFP were observed. The emission ratio (acceptor/donor) was increased 2-fold (p < 0.05) in cells expressing Ht31.ECFP and RII.EYFP compared with cells expressing Ht31P.ECFP, the inactive form of Ht31, and RII.EYFP. These results provide the first in vivo demonstration of RII/Ht31 interaction in living cells and confirm previous in vitro findings of RII/Ht31 binding. Using surface plasmon resonance, we also showed that the green fluorescent protein tags did not significantly alter the binding of Ht31 to RII. Thus, fluorescence resonance energy transfer can be used to directly monitor protein-protein interactions of the PKA signaling pathway in living cells.     

Quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide linkers

The fusion of different protein domains via peptide linkers is a powerful, modular approach to obtain proteins with new functions. A detailed understanding of the conformational behavior of peptide linkers is important for applications such as fluorescence resonance energy transfer (FRET)-based sensor proteins and multidomain proteins involved in multivalent interactions. To investigate the conformational behavior of flexible glycine- and serine-containing peptide linkers, we constructed a series of fusion proteins of enhanced cyan and yellow fluorescent proteins (ECFP-linker-EYFP) in which the linker length was systematically varied by incorporating between 1 and 9 GGSGGS repeats. As expected, both steady-state and time-resolved fluorescence measurements showed a decrease in energy transfer with increasing linker length. The amount of energy transfer observed in these fusion proteins can be quantitatively understood by simple models that describe the flexible linker as a worm-like chain with a persistence length of 4.5 A or a Gaussian chain with a characteristic ratio of 2.3. The implications of our results for understanding the properties of FRET-based sensors and other fusion proteins with Gly/Ser linkers are discussed.