High Fluorescence Resonance Energy Transfer Research Articles

Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins

We describe a new method for the assay of sequence-specific DNA-binding proteins in this paper. In this method, the sensitive fluorescence resonance energy transfer (FRET) technology is combined with the common DNA footprinting assay in order to develop a simple, rapid and high-throughput approach for quantitatively detecting the sequence-specific DNA-binding proteins. We named this method as exonuclease III (ExoIII) protection assay with FRET probe. The FRET probe used in this assay was a duplex DNA which was designed to contain one FRET pair in the center and two flanking protein-binding sites. During protein detection, if a target protein exists, it will bind to the two protein-binding sites of the FRET probe and thus protect the FRET pair from ExoIII digestion, resulting in high FRET. However, if the target protein does not exist, the FRET pair on the naked FRET probe will be degraded by ExoIII, resulting in low FRET. Three kinds of recombinant transcription factors including NF-κB, SP1 and p50, and the target protein of NF-κB in HeLa cell nuclear extracts, were successfully detected by the assay. This assay can be extensively used in biomedical research targeted at DNA-binding proteins.

Fibrinogen receptor antagonists induce conformational changes of the human platelet glycoprotein IIb.

Controversial results have been reported concerning the ability of fibrinogen receptor antagonists (fibans) to induce conformational changes in the fibrinogen receptor after binding to it as the initial step of fibrinogen binding and platelet activation. Platelets in citrated whole blood were stained with several pairs of anti-glycoprotein (anti-GP) IIb-directed monoclonal antibodies conjugated to phycoerythrin (PE) or indirectly labeled with Cy5. Pairs of monoclonal antibodies that induced a high-fluorescence resonance energy transfer (FRET) efficiency served as tools to detect activation-dependent changes of GP IIb after addition of adenosine diphosphate and several fibans. Using the combination of the clones 5B12-PE and P2-biotin/SA-Cy5, a concentration-dependent alteration of the GP IIb conformation was observed after addition of tirofiban, eptifibatide, and lotrafiban. Magnitude and kinetics differed among the investigated substances. The newly developed FRET assay allows the direct investigation of conformational changes of GP IIb after addition of platelet agonists or receptor ligands, as shown for three fibans.

A high-throughput method for development of FRET-based indicators for proteolysis

SCAT3 is a fluorescence resonance energy transfer (FRET)-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3-sensitive linker, and an enhanced yellow fluorescent protein with efficient maturation property (Venus). Despite its considerable promise, however, greater responsivity of fluorescence to the proteolysis has been desired for better understanding of spatio-temporal pattern of the activation of caspase-3 during apoptosis. In the present study, the length of linker regions of SCAT3 has been thoroughly optimized by use of a PCR technique. The bacterial colonies expressing the constructs were screened for high FRET efficiency using our home-made fluorescence image analyzer. The FRET signal of an improved SCAT3 changed by about tenfold during apoptotic events in mammalian cells, enabling visualization of caspase-3 activation with better spatial resolution than before. This new high-throughput method will be applicable to development and improvement of FRET-based indicators for proteolysis.

A high-throughput method for development of FRET-based indicators for proteolysis

SCAT3 is a fluorescence resonance energy transfer (FRET)-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3-sensitive linker, and an enhanced yellow fluorescent protein with efficient maturation property (Venus). Despite its considerable promise, however, greater responsivity of fluorescence to the proteolysis has been desired for better understanding of spatio-temporal pattern of the activation of caspase-3 during apoptosis. In the present study, the length of linker regions of SCAT3 has been thoroughly optimized by use of a PCR technique. The bacterial colonies expressing the constructs were screened for high FRET efficiency using our home-made fluorescence image analyzer. The FRET signal of an improved SCAT3 changed by about tenfold during apoptotic events in mammalian cells, enabling visualization of caspase-3 activation with better spatial resolution than before. This new high-throughput method will be applicable to development and improvement of FRET-based indicators for proteolysis.

In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine.

The investigation into the intracellular mechanisms for gene expression has acquired great impetus for the improvement of the transfection efficiency by a non-viral gene delivery system. Intracellular trafficking as well as release of plasmid DNA (pDNA) complexed with polycations, including linear and branched polyethylenimine (LPEI, BPEI) and poly(L-lysine) (PLL), were explored under confocal microscopy using fluorescence resonance energy transfer (FRET) between a pair of donor-acceptor fluorescent dyes (fluorescein and Cy3) tagged on a single pDNA molecule. pDNA complexed with LPEI underwent a rapid escape from the endosomes, spreading uniformly into the cytoplasm with a substantial decrease in FRET efficiency due to the disintegration of LPEI/pDNA polyplex structure. pDNA complexed with BPEI also achieved a rapid escape from the endosomes. Nevertheless, the pDNA retained high FRET efficiency even after 24 h, indicating an appreciable stability of the BPEI/pDNA polyplex to keep pDNA in a condensed state. In the PLL/pDNA polyplexes, neither endosome escape nor pDNA decondensation was observed. These intracellular characteristics of polyplexes showed a clear correlation to their gene transfection efficiency: The LPEI/pDNA revealed a considerably higher and faster gene expression compared with BPEI/pDNA. Atomic force microscopy revealed that BPEI induced more effective condensation of pDNA than LPEI, being consistent with restricted cytoplasmic release of complexed pDNA. Fast endosomal escape and subsequent smooth disintegration of LPEI/pDNA in the cytoplasm are likely to be determining factors for the excellent transfection efficiency of this polyplex system. These properties may be particularly beneficial to achieve appreciably high gene expression in a prompt manner.

Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes.

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3/FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3/FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C = 0.64). A different hierarchical level of molecular proximity between Kv1.3/FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E = 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.

Binding of Thermomyces (Humicola) lanuginosa lipase to the mixed micelles of cis-parinaric acid/NaTDC.

The binding of Thermomyces lanuginosa lipase and its mutants [TLL(S146A), TLL(W89L), TLL(W117F, W221H, W260H)] to the mixed micelles of cis-parinaric acid/sodium taurodeoxycholate at pH 5.0 led to the quenching of the intrinsic tryptophan fluorescence emission (300-380 nm) and to a simultaneous increase in the cis-parinaric acid fluorescence emission (380-500 nm). These findings were used to characterize the Thermomyces lanuginosa lipase/cis-parinaric acid interactions occurring in the presence of sodium taurodeoxycholate. The fluorescence resonance energy transfer and Stern-Volmer quenching constant values obtained were correlated with the accessibility of the tryptophan residues to the cis-parinaric acid and with the lid opening ability of Thermomyces lanuginosa lipase (and its mutants). TLL(S146A) was found to have the highest fluorescence resonance energy transfer. In addition, a TLL(S146A)/oleic acid complex was crystallised and its three-dimensional structure was solved. Surprisingly, two possible binding modes (sn-1 and antisn1) were found to exist between oleic acid and the catalytic cleft of the open conformation of TLL(S146A). Both binding modes involved an interaction with tryptophan 89 of the lipase lid, in agreement with fluorescence resonance energy transfer experiments. As a consequence, we concluded that TLL(S146A) mutant is not an appropriate substitute for the wild-type Thermomyces lanuginosa lipase for mimicking the interaction between the wild-type enzyme and lipids.

Refolding of triosephosphate isomerase in low-water media investigated by fluorescence resonance energy transfer.

The refolding and reassociation of rabbit muscle triosephosphate isomerase (TPI) monomers unfolded with guanidine hydrochloride (GdnHCl) was studied in aqueous media and in reverse micelles (RM) formed with hexadecyltrimethylammonium bromide and n-octane/hexanol. Fluorescence resonance energy transfer (FRET) studies were carried out using TPI labeled at Cys-217 with 5-((2-((iodoacetyl)-amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) and TPI labeled at one of the free amino groups with fluorescein 5'-isothiocyanate (FITC). Efficient FRET between monomers of AEDANS-TPI and FITC-TPI in aqueous media was detected 2-3 min after denaturant dilution and remained constant for hours. The distance between AEDANS and FITC in a labeled, renatured hetero-TPI dimer calculated from FRET results was 48 A, in reasonable agreement with estimates based on the crystal structure of TPI. In RM, recovery of enzyme activity during renaturation correlates with the appearance of a high-intrinsic fluorescence intermediate believed to be a partially folded monomer (Fernández-Velasco et al., 1995). Nevertheless, when AEDANS- and FITC-labeled monomers were mixed in RM, FRET occurred as soon as GdnHCl was diluted (FRET efficiency = 0.36), preceding the changes in TPI intrinsic fluorescence and reactivation. Thereafter, the efficiency of FRET increased during the next hour up to approximately 0.50, where it remained after 24 h, when 80% of the enzyme activity was recovered. The high initial FRET seen in RM could indicate the formation of an inactive dimer within the first minutes after denaturant dilution. The further increase in FRET observed over the next hour could reflect conformational rearrangements of the protein and transition from the inactive to the active dimer.