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.

Oxidation of unsaturated phospholipids in membrane bilayer mixtures is accompanied by membrane fluidity changes.

Steady-state and time-resolved fluorescence spectroscopy has been used to obtain information on oxidation processes and associated dynamical and structural changes in model membrane bilayers made from single unilamellar vesicles (SUV's) of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) mixed with increasing amounts of 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC). The highly unsaturated arachidonoyl chain containing four double bonds is prone to oxidation. Lipid oxidation was initiated chemically by a proper oxidant and could be followed on line via the fluorescence changes of an incorporated fluorescent lipophilic fatty acid: 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BP-C11). The oxidation rate increases with an increasing amount of SAPC. Size measurements of different SUV's incorporated with a trace amount of a phosphatidylcholine analogue of BP-C11 using fluorescence correlation spectroscopy have demonstrated that an increase of lipid unsaturation results in smaller sized SUV's and therefore to a larger curvature of the outer bilayer leaflet. This suggests that the lipid-lipid spacing has increased and that the unsaturated fatty acyl chains are better accessible for the oxidant. Oxidation results in some characteristic physical changes in membrane dynamics and structure, as indicated by the use of specific fluorescence probes. Fluorescence measurements of both dipyrenyl- and diphenylhexatriene-labelled PC introduced in non-oxidised and oxidised DOPC-SAPC membranes clearly show that the microfluidity (local fluidity at the very site of the probes) significantly decreases when the oxidised SAPC content increases in the lipid mixture. A similar effect is observed from the lateral diffusion experiments using monopyrenyl PC in the same membrane systems: the lateral diffusion is distinctly slower in oxidised membranes.

Antigen-antibody interactions: binding studies with fluorescence and surface plasmon resonance exemplified by acid-traseolide as antigen.

The interaction between the musk fragrance acid-traseolide and monoclonal antibodies (mAB) generated against this odorant has been investigated with two different techniques. Fluorescence spectroscopy was used to study the quenching of tryptophan fluorescence of the antibody upon binding acid-traseolide. This spectroscopic approach is based on measurements under equilibrium conditions. The second technique exploited the surface plasmon resonance (SPR) phenomenon. The acid-traseolide was immobilized in the surface matrix and upon presenting mAB changes in SPR were recorded in real time during the association reaction. The SPR approach can be considered as a kinetic method. Although having a different origin, both methods lead to comparable equilibrium dissociation constants (Kd). However, the results obtained with fluorescence spectroscopy were more accurate and reproducible. Not only the association of acid-traseolide with antibody was evaluated, also Fab fragment and peptide (H3-peptide) mimicking the heavy chain CDR3 of this antibody were included in this study. The Kd-values, determined by both methods, increase in the order mAB < Fab < H3-peptide because of diminishing recognition.

Time-resolved fluorescence anisotropy of HIV-1 protease inhibitor complexes correlates with inhibitory activity.

The tryptophan time-resolved fluorescence intensity and anisotropy of the HIV-1 protease dimer is shown to be a quick and efficient method for the conformational characterization of protease inhibitor complexes. Four fluorescence lifetimes were needed to adequately describe the fluorescence decay of the two tryptophan residues, W6 and W42, per protease monomer. As a result of the wavelength dependence of the respective amplitudes, the 2.06 ns and the 4.46 ns decay constants were suggested to be the intrinsic fluorescence lifetimes of the more solvent-exposed W6 and the less exposed W42 residues, respectively. Analysis of the fluorescence anisotropy decay yielded a short correlation time of 250 ps corresponding to local chromophore motions, and a long correlation time of 12.96 ns resulting from overall rotation of the protease enzyme. Fluorescence lifetimes and rotational correlation times changed when inhibitors of the HIV-1 protease were added. The effects of 11 different inhibitors including statine-derived, hydroxyethylamine-derived, and 2 symmetrical inhibitors on the protease fluorescence dynamics were investigated. Inhibitor binding is shown to induce an increase of the mean fluorescence lifetime taumean, an increase of the short rotational correlation time phi1, as well as a decrease of the long rotational correlation time phi2. The mean rotational correlation time phimean was identified as the global dynamic parameter for a given molecular complex, which correlates with the inhibitor dissociation constant Ki, and therefore with the activity of the inhibitor.

Time-resolved fluorescence investigations of the interaction of the voltage-sensitive probe RH421 with lipid membranes and proteins.

Fluorescence lifetimes and fluorescence anisotropy decays of the voltage-sensitive styryl-pyridinium dye RH421 have been measured in the presence of dimyristoylphosphatidylcholine vesicles, the water soluble enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco), and Na+,K(+)-ATPase-containing membrane fragments. The effect of an intramembrane electric field on the photophysical properties of the dye was investigated by the binding of the hydrophobic ion tetraphenylborate (TPB) to the membrane. TPB was found to significantly increase the average fluorescence lifetime and the order of membrane-bound dye. The increase in fluorescence lifetime is consistent with reorientation of the dye further into the membrane interior. The increase in order may be attributed to an electric field-induced alignment of the dye molecules. From the values of the rotational diffusion constant experimentally determined, the expected response time of the dye to an applied electric field can be calculated for a reorientational mechanism to be on the order of tens of nanoseconds. Experiments with rubisco showed that the dye interacts strongly with the protein. In this case, the dye is so tightly bound that it has almost no independent motion and rotates virtually solely with the protein. The rate of rotational motion of the dye in the presence of Na+,K(+)-ATPase-containing membrane fragments is similar to that in pure lipid membranes. The order parameter of the dye in the Na+,K(+)-ATPase membrane fragments is close to the maximum value. This is most probably due to the high density of protein molecules, which restricts the range of motion of the dye.

Fluorescence dynamics of staphylococcal nuclease in aqueous solution and reversed micelles.

The dynamical fluorescence properties of the sole tryptophan residue (Trp-140) in Staphylococcus aureus nuclease (EC 3.1.31.1) have been investigated in aqueous solution and reversed micelles composed of either sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane or cetyltrimethylammonium chloride (CTAC) in isooctane/hexanol (12:1 by volume). The fluorescence decay of nuclease in the different environments can be described by a trimodal distribution of fluorescence lifetimes at approx. 0.5, 1.5 and 5.0 ns. The relative amplitudes depend on the environment. For pH 9.0 solutions the contribution of the two shortest lifetime components in the distribution is largest for AOT and smallest for CTAC reversed micelles. There is reasonable agreement between the average fluorescence lifetime and the fluorescence quantum efficiency confirming a significant fluorescence quenching in AOT reversed micelles. Fluorescence anisotropy decay revealed that the tryptophan environment in aqueous nuclease solutions is rigid on a nanosecond timescale. When nuclease was entrapped into reversed micelles the tryptophan gained some internal flexibility as judged from the distinct presence of a shorter correlation time. The longer correlation time reflected the rotational properties of the protein-micellar system. Modulation of the overall charge of nuclease (isoelectric point pH 9.6) by using buffer of pH 9.0 and pH 10.4, respectively, and of the size of empty micelles by selecting two values of the water to surfactant molar ratio, had only a minor effect on the rotational properties of nuclease in the positively charged reversed micelles. Encapsulation of nuclease in anionic reversed micelles resulted in the development of protein bound to aggregated structures which are immobilised on a nanosecond timescale. According to far UV circular dichroism results the secondary structure of nuclease only followed the already published pH-dependent changes. Encapsulation had no major effect on the overall secondary structure.

Static and dynamic studies of the potential-sensitive membrane probe RH421 in dimyristoylphosphatidylcholine vesicles.

The dynamics of the potential-sensitive styryl dye RH421 in dimyristoylphosphatidylcholine vesicles have been investigated above and below the main phase transition temperature using iodine-laser temperature-jump relaxation spectrophotometry and time-resolved fluorescence lifetime measurements. Equilibrium fluorescence titrations have shown that the affinity of the dye for the membrane is much higher in the liquid-crystalline state than in the gel state. The interaction can be described by either a partition or a binding model and a theory is presented providing a relation between these two approaches. In the liquid-crystalline state bound dye exhibits steady-state fluorescence relaxation processes in the submicrosecond and millisecond time range following a temperature jump. Time-resolved fluorescence measurements show a variation in the fluorescence lifetime across the emission spectrum, suggesting an excited-state process occurring on the subnanosecond time scale. These processes are most likely related to dye and/or lipid reorientation following the temperature jump or excitation pulse. Temperature-dependent changes in the fluorescence excitation spectrum of bound dye suggest that the dye exists in at least two different sites within the membrane.