Contributions of fluorescence techniques to understanding G protein-coupled receptor dimerisation

G protein-coupled receptors (GPCRs) are the largest class of eukaryotic cell-surface receptors and, over the last decade, it has become clear that they are capable of dimerisation. Whilst many biochemical and biophysical approaches have been used to study dimerisation, fluorescence techniques, including Förster resonance energy transfer and single molecule fluorescence, have been key players. Here we review recent contributions of fluorescence techniques to investigate GPCR dimers, including dimerisation in cell membranes and native tissues, the effect of ligand binding on dimerisation and the kinetics of dimer formation and dissociation. The challenges of studying multicomponent membrane protein systems have led to the development and refinement of many fluorescence assays, allowing the functional consequences of receptor dimerisation to be investigated and individual protein molecules to be imaged in the membranes of living cells. It is likely that the fluorescence techniques described here will be of use for investigating many other multicomponent membrane protein systems.     

Maximum likelihood method for the analysis of time-resolved fluorescence decay curves

The usefulness of fluorescence techniques for the study of macromolecular structure and dynamics depends on the accuracy and sensitivity of the methods used for data analysis. Many methods for data analysis have been proposed and used, but little attention has been paid to the maximum likelihood method, generally known as the most powerful statistical method for parameter estimation. In this paper we study the properties and behavior of maximum likelihood estimates by using simulated fluorescence intensity decay data. We show that the maximum likelihood method provides generally more accurate estimates of lifetimes and fractions than does the standard least-squares approach especially when the lifetime ratios between individual components are small. Three novelties to the field of fluorescence decay analysis are also introduced and studied in this paper: a) discretization of the convolution integral based on the generalized integral mean value theorem: b) the likelihood ratio test as a tool to determine the number of exponential decay components in a given decay profile; and c) separability and detectability indices which provide measures on how accurately, a particular decay component can be detected. Based on the experience gained from this and from our previous study of the Padé-Laplace method, we make some recommendations on how the complex problem of deconvolution and parameter estimation of multiexponential functions might be approached in an experimental setting. Offprint requests to: F. G. Prendergast     

Solid state fluorescence of lyophilized proteins

Fluorescence spectroscopy has been used to measure changes in the tertiary structure of proteins in the solution state. The sensitivity of fluorescence to the protein tryptophan environment has made it a useful tool for studying protein conformation and stability. Using fluorescence spectroscopy to probe structural alterations in lyophilized proteins has been limited due to technical challenges and overwhelming background light scattering. We have investigated the possibility of analyzing lyophilized proteins using the Cary-Eclipse spectrofluorometer by monitoring the fluorescence of the protein therapeutic after subjecting the lyophilized cake to heat-induced accelerated degradation. We have been able to obtain reproducible fluorescence spectra, detecting possible structural changes under these conditions. Fluorescence and circular dichroism spectroscopic analyses of the reconstituted proteins indicated that changes in fluorescence intensities observed in the solid state could be correlated to that in solution and to possible tertiary structural changes. Size exclusion chromatography analysis of protein Y subject to accelerated degradation showed a correlation between decreasing fluorescence intensity and increasing protein Y tetramer in solution, consistent with long-term stability. This suggests that solid state, intrinsic protein fluorescence measurements using the Cary-Eclipse holder may be feasible for long-term stability studies and formulation development.     

Fluorescence correlation spectroscopy in the nanosecond time range: Photon antibunching in dye fluorescence

Possibilities for the use of fluorescence correlation spectroscopy in the nanosecond time range are demonstrated. The experiment is based on a cw argon ion laser, a microfluorimeter, two photon detectors, and a time-to-analog converting system. Experiments using solutions of rhodamine 6G and pyronine G in water at concentrations of about 20 molecules per sample volume are reported. The photon anticorrelation component decaying with a time constant close to the excited state lifetime was observed.     

Two-photon thermal bleaching of single fluorescent molecules

We have studied the fluorescence emission by two-photon excitation of four dyes widely used for bioimaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level. The single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output until a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution indicating that the observed behavior is not due to photobleaching. Moreover, the bleaching time decreases with the glass substrate temperature reaching a vanishing nonmeasurable value for a limiting temperature whose value is found in the same series as for the bleaching time, from the lowest to the highest temperature respectively. The observed bleaching shows a clear correlation to the amount of absorbed power not reirradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon absorption of the single dyes as confirmed also by numerical simulations.     

Molecular structure,vibrational spectra and HOMO,LUMO analysis of 4-piperidone by density functional theory and ab initio Hartree–Fock calculations

Vibrational frequencies and geometrical parameters of 4-piperidone (4-PID) in the ground state have been calculated by using the Hartree–Fock (HF) and density functional methods (B3LYP) with 6-311++G(d,p) and 6-311+G(3df,2p) basis sets. These methods are proposed as a tool to be applied in the structural characterisation of 4-PID (C₅H₉NO). The title molecule has C _s point group symmetry, thus providing useful support in the interpretation of experimental IR and Raman data. The DFT-B3LYP/6-311+G(3df,2p) calculations have been found more reliable than the ab initio HF/6-311++G(d,p) calculations for the vibrational study of 4-PID. The calculated highest occupied molecular orbital and lowest unoccupied molecular orbital energies show that charge transfer occurs within the molecule. The theoretical spectrograms for FT-IR and FT-Raman spectra of the title molecule have been constructed.     

Statistical accuracy in fluorescence fluctuation experiments

The mathematical expression of the signal to noise ratio in fluorescence fluctuation experiments is derived for arbitrary sample profiles and for any mechanism of translational motion, and experimentally verified. The signal to noise ratio depends on the mean count rate per particle per dwell time, the mean number of particles per sample volume, time characteristics of the correlation function, sample profile characteristics, and the data collection time. Statistical accuracy of the third order moment of fluorescence intensity fluctuations is also studied. The optimum concentration for the third order moment analysis is about one particle per sample volume. Received: 13 February 1996 / Accepted: 20 September 1996     

The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue

Hydrophobic sites on the surface of protein molecules are thought to have important functional roles. The identification of such sites can provide information about the function and mode of interaction with other cellular components. While the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, strong intrinsic quenching of Nile red and ANSA dye fluorescence is observed on binding to a cytochrome c('). Fluorescence quenching is also observed to take place in the presence of a variety of other biologically important molecules which can compromise the quantitative determination of binding constants. Absorption difference spectroscopy is shown not to be sensitive to the presence of fluorescence quenchers but sensitive enough to measure binding constants. The dye BPB is shown to bind to the same hydrophobic sites on proteins as polarity-sensitive fluorescence probes. The absorption spectrum of BPB is also observed to be polarity sensitive. A binding constant of 3x10(6)M(-1) for BPB to BSA has been measured by absorption difference spectroscopy. An empirical correlation is observed between the shape of the absorption difference spectrum of BPB and the polarity of the environment. The results indicate that absorption difference spectroscopy of BPB provides a valuable supplement to fluorescence for determining the presence of hydrophobic sites on the surface of proteins as well as a method for measuring binding constants.     

Small Molecule Diffusion into Swelling Iota-Carrageenan Gels: A Fluorescence Study

Abstract

Small molecule diffusion into Iota-Carrageenan gel was studied by using steady-state fluorescence (SSF) technique. Pyranine, dissolved in water was used as fluorescence probe. Fluorescence emission intensity, I_p, and scattered light intensity, I_sc, were monitored to study diffusion and swelling processes at various temperatures respectively. Fickian and Li-Tanaka models were elaborated to produce diffusion, D, and collective diffusion, D ₀, coefficients. Diffusion and swelling activation energies were also obtained and found to be 20.5 kj mol^?1 and 28.2 kj mol^?1, respectively.     

The ELLIPS suite of macromolecular conformation algorithms

This paper describes a series of four programmes for the PC based on ellipsoidal representations of macromolecular shape in solution using Universal shape functions. ELLIPS1 is based on simple ellipsoid of revolution models (where two of the three axes of the ellipsoid are fixed equal to each other). If the user types in a value for a shape function from sedimentation or other types of hydrodynamic measurement, it will return a value for the axial ratio of the ellipsoid. ELLIPS2 is based on the more general triaxial ellipsoid with the removal of the restriction of two equal axes. The user enters the three semi-axial dimensions of the molecule or the equivalent two axial ratios and ELLIPS2 returns the value of all the hydrodynamic shape functions. It also works of course for ellipsoids of revolution. ELLIPS3 and ELLIPS4 do the reverse of ELLIPS2, that is they both provide a method for the unique evaluation of the triaxial dimensions or axial ratios of a macromolecule (and without having to guess a value for the so-called „hydration”) after entering at least three pieces of hydrodynamic information: ELLIPS3 requires EITHER the intrinsic viscosity with the second virial coefficient (from sedimentation equilibrium, light scattering or osmometry) and the radius of gyration (from light or x-ray scattering) OR the intrinsic viscosity with the concentration dependence term for the sedimentation coefficient and the (harmonic mean) rotational relaxation time from fluorescence depolarisation measurements. ELLIPS4 evaluates the tri-axial shape of a macromolecule from electro-optic decay based Universal shape functions using another Universal shape function as a constraint in the extraction of the decay constants. Accepted: 1 November 1996     

Padé-Laplace method for the analysis of time-resolved fluorescence decay curves

The interpretation of fluorescence intensity decay times in terms of protein structure and dynamics depends on the accuracy and sensitivity of the methods used for data analysis. The are many methods available for the analysis of fluorescence decay data, but justification for choosing any one of them is unclear. In this paper we generalize the recently proposed Padé-Laplace method [45] to include deconvolution with respect to the instrument response function. In this form the method can be readily applied to the analysis of time-correlated single photon counting data. By extensive simulations we have shown that the Padé-Laplace method provides more accurate results than the standard least squares method with iterative reconvolution under the condition of closely spaced lifetimes. The application of the Padé-Laplace method to several experimental data sets yielded results consistent with those obtained by use of the least squares analysis. Offprint requests to: F. G. Prendergast     

Direct observation of the distribution of fluorescent probes in phosphatidylcholine/cholesterol vesicles using flow microfluorometry

The technique of flow microfluorometry has been extended to the study of small lipid complexes to assess either the lipid (hydrophobic) or aqueous (hydrophilic) compartments of selected natural or model membrane systems. sn-1-Palmitoyl-sn-2-oleoyl-phosphatidylcholine/cholesterol unilamellar vesicles, averaging 268 nm in diameter and containing varying concentrations of the synthetic lipophile probe, sn-1-palmitoyl-sn-2-12-[N-4-nitrobenzo-2-oxa-1,3-diazole]-aminocaproyl-phosphatidylcholine (NBD-PC), were analyzed using an Ortho Series 50-H Cytofluorograf and an Ortho 2150 computer system. NBD-labeled vesicles were analyzed for green fluorescence and the intensity of scattered light, the later being analyzed both at low angle (2–5°) and at 90° to the incident beam. At the high amplification required for vesicle detection, background signals from the sheath buffer, nonspecific laser light, and electronic noise were observed. However, this background noise signal was removed by appropriately setting a discriminator window. Profiles of signals falling within this region were then constructed. For the settings selected, more than 98% of data recorded could be attributed to observations on vesicles. Size information from the intensity of scattered light was obtained by comparison of the sample with fluorescent microspheres after correcting for the particle-scattering function difference between hollow and solid spheres and for refractive index differences. Additionally, cytograms and profiles were constructed for vesicles containing 5 m 6-carboxyfluorescein, 3′,6′-dihydroxy-3-oxospiro(isobenzofuran-1 (3H),9′-(9H)xanthen)-6-carboxylic acid, trapped in the aqueous core. Thus, the utility of flow microfluorometry has been extended to much smaller particle populations than studied previously by this technique. It has significant potential for studying several important properties of selected populations of vesicles and lipoproteins including (i) the size and fluorescence distribution of particles, (ii) the equilibrium distribution of probes among different size populations and among different domains within populations, (iii) the time dependence of probe transfer from a specific labeled population to a specific unlabeled population, (iv) the time dependence of vesicle fusion (combining aqueous compartments), and (v) sorting particles which are labeled differently.     

Characterization of a fluorophore binding RNA aptamer by fluorescence correlation spectroscopy and small angle X-ray scattering

Using fluorescence correlation spectroscopy (FCS), we have established an in vitro assay to study RNA dynamics by analyzing fluorophore binding RNA aptamers at the single molecule level. The RNA aptamer SRB2m, a minimized variant of the initially selected aptamer SRB-2, has a high affinity to the disulfonated triphenylmethane dye sulforhodamine B. A mobility shift of sulforhodamine B after binding to SRB2m was measured. In contrast, patent blue V (PBV) is visible only if complexed with SRB2m due to increased molecular brightness and minimal background. With small angle X-ray scattering (SAXS), the three-dimensional structure of the RNA aptamer was characterized at low resolution to analyze the effect of fluorophore binding. The aptamer and sulforhodamine B-aptamer complex was found to be predominantly dimeric in solution. Interaction of PBV with SRB2m led to a dissociation of SRB2m dimers into monomers. Radii of gyration and hydrodynamic radii, gained from dynamic light scattering, FCS, and fluorescence cross-correlation experiments, led to comparable conclusions. Our study demonstrates how RNA-aptamer fluorophore complexes can be simultaneously structurally and photophysically characterized by FCS. Furthermore, fluorophore binding RNA aptamers provide a tool for visualizing single RNA molecules.     

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single-dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.     

Catecholamine fluorescence in the pituitary of the eel,Anguilla anguilla,with special reference to its variation during background adaptation

Summary In the neuro-intermediate lobe (NIL) of the eel, Anguilla anguilla, a specific formaldehyde-induced fluorescence, indicating a catecholamine (CA) innervation, has been demonstrated in the neural lobe processes. Microspectrofluorimetric analyses and pharmacological treatments indicate noradrenaline or dopamine or both to be responsible for the fluorescence.The fluorescence in the NIL has displayed a definite tendency toward variation during the adaptation to a white and to a black background. The highest amounts of fluorescence were generally found in animals adapted to a black background, especially when adapted for a rather long period, and in animals recently transferred to a white background. The lowest amounts of fluorescence were generally found in animals adapted to a white background.This and the result of injections of CA-depleting drugs suggest that the monoaminergic nerves are active when the animal is on a white background, inhibiting the MSH release directly or indirectly or both, or in co-operation with other factors.Specific green fluorescent structures were also found in other parts of the neural lobe supplying the pars distalis.In some pharmacologically untreated specimens and in animals treated with CA-depleting drugs, the intermedia cells fluoresced. Microspectrofluorimetric analyses indicated that this fluorophore was not a CA.We wish to express our sincere thanks to Miss Ingrid Carlsen for excellent technical assistance, Mr. Lajos Erdös for the photography and the technical staff of the Department of Histology in Lund. We are also indepted to Dr. Anders Björklund for valuable discussion and advice.Supported by grants from the Swedish Natural Science Research Council, the University of Lund, and the Royal Physiographic Society of Lund.     

Enabler for process analytical technology implementation in Pichia pastoris fermentation: Fluorescence‐based soft sensors for rapid quantitation of product titer

Rapid quantitation of product titer is a critical input for control of any bioprocess. This measurement, however, is marred by the myriad components that are present in the fermentation broth, often requiring extensive sample pretreatment before analysis. Spectroscopy techniques such as fluorescence spectroscopy are widely recognized as potential monitoring tools. Here, we investigate the possibility of using fluorescence of the culture supernatant as a potential at‐line monitoring tool to measure the concentration of a recombinant therapeutic protein expressed in a Pichia pastoris fed‐batch fermentation. We propose an integrated method wherein both the target protein and total protein concentrations are predicted using intrinsic riboflavin fluorescence and extrinsic fluorescence, respectively. The root mean square error for estimating the concentrations of the target protein (using riboflavin fluorescence) and total protein (using extrinsic fluorescence) have been estimated to be <0.1 and <0.2, respectively. The proposed approach has been validated for two different biotherapeutic products, human serum albumin and granulocyte colony stimulating factor, that were expressed using Mut⁺ and Mut^s strains of P. pastoris, respectively. The proposed approach is rapid (1 min analysis time, 10 min total with at line sampling) and thus could be a significant enabler for process analytical technology implementation in Pichia fermentation.     

International study of factors affecting human chromosome translocations

Chromosome translocations in peripheral blood lymphocytes of normal, healthy humans increase with age, but the effects of gender, race, and cigarette smoking on background translocation yields have not been examined systematically. Further, the shape of the relationship between age and translocation frequency (TF) has not been definitively determined. We collected existing data from 16 laboratories in North America, Europe, and Asia on TFs measured in peripheral blood lymphocytes by fluorescence in situ hybridization whole chromosome painting among 1933 individuals. In Poisson regression models, age, ranging from newborns (cord blood) to 85 years, was strongly associated with TF and this relationship showed significant upward curvature at older ages versus a linear relationship (p < 0.001). Ever smokers had significantly higher TFs than non-smokers (rate ratio (RR) = 1.19, 95% confidence interval (CI), 1.09–1.30) and smoking modified the effect of age on TFs with a steeper age-related increase among ever smokers compared to non-smokers (p < 0.001). TFs did not differ by gender. Interpreting an independent effect of race was difficult owing to laboratory variation. Our study is three times larger than any pooled effort to date, confirming a suspected curvilinear relationship of TF with age. The significant effect of cigarette smoking has not been observed with previous pooled studies of TF in humans. Our data provide stable estimates of background TF by age, gender, race, and smoking status and suggest an acceleration of chromosome damage above age 60 and among those with a history of smoking cigarettes.     

Features of the Secondary Emission Enhancement Near Plasmonic Gold Film

Plasmonic gold films (PGF) prepared by vacuum deposition of gold onto quartz slides possess unique property to enhance electromagnetic signal in the near field. Spectral tuning of PGF’s plasmon band to resonance with the electronic spectra of adsorbed molecules provides selective enhancement of fluorescence or surface-enhanced Raman scattering in the far field. Plasmon-enhanced fluorescence (PEF) of mitoxantrone (mitox) as a function of the distance between gold surface and adsorbed molecules for different polarization and incidence angle of exciting light is analyzed in this work. Spectrophotometric data reveal that probability of localized plasmon excitation in gold grains increases with growth of incidence angle for s-polarized and decrease for p-polarized excitation. This fact correlates well with oblate shape of gold particles detected by Atomic force microscope. However, the fluorescence intensity of dyes deposited at fixed distance from gold surface increase with angle of incidence of p-polarized light more noticeably than for s-polarized one. Nevertheless, the behavior of mitox PEF signal upon p-polarized laser excitation and different angle of incidence are similar in appearance to such phenomenon as selective photoelectric effect. According to this observation, the near-field interactions between plasmons and molecule as possible mechanism of PEF is discussed.     

Optical features of the fluorophore azotobactin: Applications for iron sensing in biological fluids

Siderophores are bio‐organic ligands secreted by microbes to chelate and assimilate iron to meet their metabolic requirements. Siderophores and their analogs have tremendous therapeutic and analytical potential including the use as Fe (III) biosensors; however, only few practical applications have been realized. The aim of this study was the optical and biophysical characterization of the siderophore azotobactin (Az) secreted by the nitrogen‐fixing bacteria Azotobacter vinelandii. The peptide exhibited fluorescence in the visible range. Quantum yield and lifetime in excited state were measured to ascertain the sensitivity of the molecule as a fluorescent marker in biochemical assays. Its high affinity toward iron in the ferric state was demonstrated through fluorescence emission quenching studies. The accuracy of azotobactin as biosensing tool was determined by analyzing the levels of iron in biological fluids, particularly in human serum. Furthermore, it was demonstrated that it can be encapsulated in sol–gel matrices without significant loss of its fluorescence signal, thus enabling it suitable for adaptation to optical biosensor for Fe (III).     

Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.Abbreviations dipyr₄ sn-1,2-(pyrenylbutyl) - dipyr₁₀ sn-1,2-(pyrenyldecanoyl). - DMPC dimyristoyl-phosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - PC phosphatidylcholine - PS phosphatidylserine Correspondence to: A. J. W. G. Visser