Nina V. Visser

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 (SUVs) 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 SUVs 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 SUVs 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.

Physical Interactions of the Peroxisomal Targeting Signal 1 Receptor Pex5p, Studied by Fluorescence Correlation Spectroscopy

We have studied Hansenula polymorpha Pex5p and Pex8p using fluorescence correlation spectroscopy (FCS). Pex5p is the Peroxisomal Targeting Signal 1 (PTS1) receptor and Pex8p is an intraperoxisomal protein. Both proteins are essential for PTS1 protein import and have been shown to physically interact. We used FCS to analyze the molecular role of this interaction. FCS is a very sensitive technique that allows analysis of dynamic processes of fluorescently marked molecules at equilibrium in a very tiny volume. We used this technique to determine the oligomeric state of both peroxins and to analyze binding of Pex5p to PTS1 peptides and Pex8p. HpPex5p and HpPex8p were overproduced in Escherichia coli, purified by affinity chromatography, and, when required, labeled with the fluorescent dye Alexa Fluor 488. FCS measurements revealed that the oligomeric state of HpPex5p varied, ranging from monomers at slightly acidic pH to tetramers at neutral pH. HpPex8p formed monomers at all pH values tested. Using fluorescein-labeled PTS1 peptide and unlabeled HpPex5p, we established that PTS1 peptide only bound to tetrameric HpPex5p. Upon addition of HpPex8p, a heterodimeric complex was formed consisting of one HpPex8p and one HpPex5p molecule. This process was paralleled by dissociation of PTS1 peptide from HpPex5p, indicating that Pex8p may play an important role in cargo release from the PTS1 receptor. Our data show that FCS is a powerful technique to explore dynamic physical interactions that occur between peroxins during peroxisomal matrix protein import.

Practical use of corrected fluorescence excitation and emission spectra of fluorescent proteins in Forster Resonance Energy Transfer (FRET) studies.

Corrected fluorescence excitation and emission spectra have been obtained from several enhanced variants of the green fluorescent protein (EGFP) isolated from the jellyfish Aequorea victoria, blue fluorescence protein (EBFP), cyan fluorescent protein (ECFP), EGFP and yellow fluorescent protein (EYFP–citrine) and from the red fluorescent protein (DsRed) isolated from the coral species Discosoma. The spectra are stored in a database. This report describes how the spectra can be used as templates to derive the critical transfer distance for any pair of fluorescent proteins.

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.

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.

Circular dichroism spectroscopy of fluorescent proteins

Circular dichroism (CD) spectra have been obtained from several variants of green fluorescent protein: blue fluorescent protein (BFP), enhanced cyan fluorescent protein (CFP), enhanced green fluorescent protein (GFP), enhanced yellow fluorescent protein (YFP), all from Aequorea victoria, and the red fluorescent protein from the coral species Discosoma (DsRed). We demonstrate that CD spectra in the spectral fingerprint region of the chromophore yield spectra that after normalization are not coincident with the normalized absorbance spectra of GFP, YFP and DsRed. On the other hand, the CD spectra of BFP and CFP coincide with the absorbance spectra. The resolution of absorption and CD spectra into Gaussian bands confirmed the location of the different electronic band positions of GFP and YFP as reported in the literature using other techniques. In the case of BFP and CFP the location of Gaussian bands provided information of the vibrational progression of the electronic absorption bands. The CD spectrum of DsRed is anomalous in the sense that the major CD band has a clear excitonic character. Far‐UV CD spectra of GFP confirmed the presence of the high β‐sheet content of the polypeptide chain in the three‐dimensional structure.

Tryptophan-Tryptophan Energy Migration as a Tool to Follow Apoflavodoxin Folding

Submolecular details of Azotobacter vinelandii apoflavodoxin (apoFD) (un)folding are revealed by time-resolved fluorescence anisotropy using wild-type protein and variants lacking one or two of apoFDs three tryptophans. ApoFD equilibrium (un)folding by guanidine hydrochloride follows a three-state model: native <--> unfolded <--> intermediate. In native protein, W128 is a sink for Förster resonance energy transfer (FRET). Consequently, unidirectional FRET with a 50-ps transfer correlation time occurs from W167 to W128. FRET from W74 to W167 is much slower (6.9 ns). In the intermediate, W128 and W167 have native-like geometry because the 50-ps transfer time is observed. However, non-native structure exists between W74 and W167 because instead of 6.9 ns the transfer correlation time is 2.0 ns. In unfolded apoFD this 2.0-ns transfer correlation time is also detected. This decrease in transfer correlation time is a result of W74 and W167 becoming solvent accessible and randomly oriented toward one another. Apparently W74 and W167 are near-natively separated in the folding intermediate and in unfolded apoFD. Both tryptophans may actually be slightly closer in space than in the native state, even though apoFDs radius increases substantially upon unfolding. In unfolded apoFD the 50-ps transfer time observed for native and intermediate folding states becomes 200 ps as W128 and W167 are marginally further separated than in the native state. Apparently, apoFDs unfolded state is not a featureless statistical coil but contains well-defined substructures. The approach presented is a powerful tool to study protein folding.

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.

A General Approach for Detecting Folding Intermediates from Steady-State and Time-Resolved Fluorescence of Single-Tryptophan-Containing Proteins

During denaturant-induced equilibrium (un)folding of wild-type apoflavodoxin from Azotobacter vinelandii, a molten globule-like folding intermediate is formed. This wild-type protein contains three tryptophans. In this study, we use a general approach to analyze time-resolved fluorescence and steady-state fluorescence data that are obtained upon denaturant-induced unfolding of a single-tryptophan-containing variant of apoflavodoxin [i.e., W74/F128/F167 (WFF) apoflavodoxin]. The experimental data are assembled in matrices, and subsequent singular-value decomposition of these matrices (i.e., based on either steady-state or time-resolved fluorescence data) shows the presence of three significant, and independent, components. Consequently, to further analyze the denaturation trajectories, we use a three-state protein folding model in which a folding intermediate and native and unfolded protein molecules take part. Using a global analysis procedure, we determine the relative concentrations of the species involved and show that the stability of WFF apoflavodoxin against global unfolding is ∼4.1 kcal/mol. Analysis of time-resolved anisotropy data of WFF apoflavodoxin unfolding reveals the remarkable observation that W74 is equally well fixed within both the native protein and the molten globule-like folding intermediate. Slight differences between the direct environments of W74 in the folding intermediate and native protein cause different rotameric populations of the indole in both folding species as fluorescence lifetime analysis reveals. Importantly, thermodynamic analyses of the spectral denaturation trajectories of the double-tryptophan-containing protein variants WWF apoflavodoxin and WFW apoflavodoxin show that these variants are significantly more stable (5.9 kcal/mol and 6.8 kcal/mol, respectively) than WFF apoflavodoxin (4.1 kcal/mol) Hence, tryptophan residues contribute considerably to the 10.5 kcal/mol thermodynamic stability of native wild-type apoflavodoxin.

Comparison between fluorescence correlation spectroscopy and time-resolved fluorescence anisotropy as illustrated with a fluorescent dextran conjugate

The motional properties of rhodamine green alone and conjugated to 10-kDa dextran have been studied by fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA). With FCS the translational diffusion times of the fluorescent particles can be determined, which are directly proportional to the shear viscosity as shown in aqueous solutions of different sucrose concentrations. With TRFA the rotational correlation times of the fluorescent particles can be determined. TRFA experiments in the case of fluorescent dextran reveal a distinct restricted internal motion of the fluorescent probe independent of the slower overall rotation of the polysaccharide. The fast depolarization is most likely due to internal motion and not to energy transfer between different rhodamine green molecules in the same dextran, since a higher viscosity of the solvent increases the correlation time for internal motion proportionally. FCS and TRFA yield complementary information in the sense that the correlation time for overall dextran rotation can be accurately determined from the translational diffusion coefficient.