Encapsulation into complex coacervate core micelles promotes EGFP dimerization.

Complex coacervate core micelles (C3Ms) are colloidal structures useful for encapsulation of biomacromolecules. We previously demonstrated that enhanced green fluorescent protein (EGFP) can be encapsulated into C3Ms using the diblock copolymer poly(2-methyl-vinyl-pyridinium)41-b-poly(ethylene-oxide)205. This packaging resulted in deviating spectroscopic features of the encapsulated EGFP molecules. Here we show that for monomeric EGFP variant (mEGFP) micellar encapsulation affects the absorption and fluorescence properties to a much lesser extent, and that changes in circular dichroism characteristics are specific for encapsulated EGFP. Time-resolved fluorescence anisotropy of encapsulated (m)EGFP established the occurrence of homo-FRET (Förster resonance energy transfer) with larger transfer correlation times in the case of EGFP. Together, these findings support that EGFP dimerizes whereas the mEGFP mainly remains as a monomer in the densely packed C3Ms. We propose that dimerization of encapsulated EGFP causes a reorientation of Glu222, resulting in a pKa shift of the chromophore, which is fully reversible after release of EGFP from the C3Ms at a high ionic strength.

Disentangling picosecond events that complicate the quantitative use of the calcium sensor YC3.60.

Yellow Cameleon 3.60 (YC3.60) is a calcium sensor based on Förster resonance energy transfer (FRET). This sensor is composed of a calmodulin domain and a M13 peptide, which are located in between enhanced cyan-fluorescent protein (ECFP) and the Venus variant of enhanced yellow-fluorescent protein (EYFP). Depending on the calcium concentration, the efficiency of FRET from donor ECFP to acceptor EYFP is changing. In this study, we have recorded time-resolved fluorescence spectra of ECFP, EYFP, and YC3.60 in aqueous solution with picosecond time resolution, using different excitation wavelengths. Detailed insight in the FRET kinetics was obtained by using global and target analyses of time- and wavelength-resolved fluorescence of purified YC3.60 in calcium-free and calcium-bound conformations. The results clearly demonstrate that for both conformations, there are two distinct donor populations: a major one giving rise to FRET and a minor one not able to perform FRET. The transfer time for the calcium-bound conformation is 21 ps, whereas it is in the order of 1 ns for the calcium-free conformation. Ratio imaging of acceptor and donor fluorescence intensities of YC3.60 is usually applied to measure Ca(2+) concentrations in living cells. From the obtained results, it is clear that the intensity ratio is strongly influenced by the presence of donor molecules that do not take part in FRET, thereby significantly affecting the quantitative interpretation of the results.

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs.

Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET-FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive.

5-fluorotryptophan as dual probe for ground-state heterogeneity and excited-state dynamics in apoflavodoxin.

The apoflavodoxin protein from Azotobacter vinelandii harboring three tryptophan (Trp) residues, was biosynthetically labeled with 5-fluorotryptophan (5-FTrp). 5-FTrp has the advantage that chemical differences in its microenvironment can be sensitively visualized via (19)F NMR. Moreover, it shows simpler fluorescence decay kinetics. The occurrence of FRET was earlier observed via the fluorescence anisotropy decay of WT apoflavodoxin and the anisotropy decay parameters are in excellent agreement with distances between and relative orientations of all Trp residues. The anisotropy decay in 5-FTrp apoflavodoxin demonstrates that the distances and orientations are identical for this protein. This work demonstrates the added value of replacing Trp by 5-FTrp to study structural features of proteins via (19)F NMR and fluorescence spectroscopy.

Structural changes of yellow Cameleon domains observed by quantitative FRET analysis and polarized fluorescence correlation spectroscopy.

Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand binding.

Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fischeri NAD(P)H:FMN-oxidoreductase.

The bioluminescent bacterial enzyme system NAD(P)H:FMN-oxidoreductase-luciferase has been used as a test system for ecological monitoring. One of the modes to quench bioluminescence is the interaction of xenobiotics with the enzymes, which inhibit their activity. The use of endogenous flavin fluorescence for investigation of the interactions of non-fluorescent compounds with the bacterial luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri has been proposed. Fluorescence spectroscopy methods have been used to study characteristics of endogenous flavin fluorescence (fluorophore lifetime, the rotational correlation time). The fluorescence anisotropy behaviour of FMN has been analysed and compared to that of the enzyme-bound flavin. The fluorescence characteristics of endogenous flavin of luciferase and NAD(P)H:FMN-oxidoreductase have been shown to be applicable in studying enzymes' interactions with non-fluorescent compounds.

Effect of quinones and phenols on the triple-enzyme bioluminescent system with protease.

The study addressed the effects of redox-active compounds on trypsin activity. Series of organic oxidizers (quinones) and reducers (phenols) were chosen as model redox-active compounds. Trypsin activity was quantified by bioluminescent technique. Interactions of these compounds with trypsin were studied by fluorescent and light absorption methods. Luminescence intensity decay constants in the reduced nicotinamidadeninedinucleotide (NADH): flavinmononucleotide (FMN)-oxidoreductase (R)-luciferase (L)-trypsin (T) (R + L + T) triple-enzyme system were calculated and compared in the presence of different concentrations of quinones and phenols. The triple-enzyme system was shown to be sensitive to quinones and not sensitive to phenols. It has been found that the effects produced by quinones on the coupled enzyme system (R + L) and on the trypsin molecule (T) are not related. The conclusions were extrapolated to the properties of other proteases and antiproteases.

Interaction of aromatic compounds with Photobacterium leiognathi luciferase: fluorescence anisotropy study.

The time-resolved and steady-state fluorescence techniques were employed to elucidate possible interactions of four aromatic compounds (anthracene, POPOP, MSB and 1,4-naphthalendiol) with bacterial luciferase. Fluorescence spectra and fluorescence anisotropy decays of these compounds were studied in ethanol, water-ethanol solutions and in the presence of bacterial luciferase. Shifts of fluorescent spectra and differences in rotational correlation times are interpreted in terms of weak (hydrophobic) interactions of the molecules with the enzyme. These interactions suggest the feasibility of intermolecular energy transfer by an exchange resonance mechanism with a collision-interaction radius as a way of excitation of these compounds in the reaction catalysed by bacterial luciferase.

Estimation of energy of the upper electron-excited states of the bacterial bioluminescent emitter.

The hypothesis of activity of the upper electron-excited states of the bacterial bioluminescent emitter was verified using dye molecules as foreign energy acceptors. Six compounds were selected having fluorescent state energies ranging from 25,700 to 32,000 cm(-1) (anthracene, pyrene, 1.4-bis(5-phenyloxasol-2-yl)benzene (POPOP), p-bis(o-methylstyryl)benzene (MSB), 2-methoxy-naphtalene, p-terphenyl), exceeding that of the bioluminescent emitter (22,000 cm(-1)). Their absorption spectra do not overlap with the bioluminescence spectrum; the trivial light absorption and the intermolecular resonance S-S energy transfer were excluded. Bacterial bioluminescent spectra of the coupled enzyme system NADH:FMN-oxidoreductase-luciferase in the presence of MSB were presented as an example. The weak sensitized fluorescence of MSB was registered. The results obtained have confirmed the activity of the energetic precursor in the bacterial bioluminescence. Its energy can be located in the interval of 26,000-27,000 cm(-1).