Fluorescence polarization studies of squid giant axons stained with N-methylanilinonaphthalenesulfonates.

The polarized components of the extrinsic fluorescence of squid giant axons stained with 2,6-MANS or 1,8-MANS were studied. The polarization properties of the fluorescence changes associated with voltage-clamp pulses were found to be very different from those of the static fluorescence, supporting the notion that the optical changes involve highly oriented membrane adsorbed fluorophores. The theoretical expectations according to this hypothesis are discussed in detail. The experimental results are in good agreement with the theory assuming that possible probes reorientations are soley due to the action of the applied electric field upon the probes electric dipole. The quantitative analysis of the data for 2,6-MANS provides a fairly accurate determination of the orientation of the membrane bound 2,6-MANS molecules responsible for the fluorescence changes. Such orientation appears to be independent of the membrane face exposed to staining. The data for 1,8-MANS indicate a very different orientation of this isomer. The results suggest a profitable use of extrinsic fluorophores for studies of the structural organization of nerve membranes.     

A comparative analysis of extrinsic fluorescence in nerve membranes and lipid bilayers

Changes in extrinsic fluorescence intensity, associated with step changes in membrane potential, have been studied in intracellularly or extracellularly stained squid axons, and in lipid bilayers, using six different aminonaphthalene dyes: 1,8-TNS; 2,6-TNS; 1,8-MANS; 2,6-MANS; 2,6-ANS and NPN. In all preparations the optical signals were found to be roughly proportional to the voltage applied. All signals had a very fast initial component, which was followed in some case by a slower change in the same direction. The slow component was observed only in intracellularly stained axons, and not for all chromophores studied. 1,8-TNS, 1,8-MANS and 2,6-MANS yielded the largest fluorescence signals in all preparations. The sign of these signals was independent of the type of membrane studied. However, the fluorescence changes of 2,6-MANS were opposite to those of 1,8-TNS and 1,8 MANS. Staining of both sides of the axolemma with 1,8-MANS or 2,6-MANS showed that these dyes yield larger signals when applied to the extracellular face. The changes in fluorescence light intensity of 2,6-TNS, 2,6-ANS and NPN were smaller and their sign depended on the membrane preparation studied. The comparison of the extrinsic fluorescence signals from the nerve membrane and the phosphatidylcholine bilayer suggests strong similarities between the basic structures of the two systems. The variety of observed signals cannot be easily interpreted in terms of changes in membrane structure. A possible alternative interpretation in terms of electrically induced displacements, rotations and changes in partition coefficient of bound chromophores, is discussed.Abbreviations 1,8-TNS 1-toluidinonaphthalene-8-sulfonate, and similarly, 2,6-TNS - 1,8-MANS 1-N-methylanilinonaphthalene-8-sulfonate, and similarly, 2,6-MANS - 1,8-ANS 1-anilinonaphthalene-8-sulfonate, and similarly, 2,6-ANS - NPN N-phenyl-1-naphthylamine     

Orientation and rotational freedom of fluorescent probes in lecithin bilayers.

The fluorescence polarization properties of lecithin bilayers stained with 2,6-MANS and 1,8 ANS under applied potential steps have been studied. The fluorescence signal components of both dyes were found to have different sign and relative amplitude, suggesting that 1,8-ANS and 2,6-MANS behave differently when bound to black lipid membranes. In order to determine the location and the extent of rotational brownian motions of the bound chromophores, the experimental data were analyzed by using a simplified physico-mathematical model. According to it 2,6-MANS appears to have a ratio rho/tau higher than 1.8-ANS (rho being the rotational relaxation of in plane rotations and tau the lifetime of the excited singlet state of the bound molecules), suggesting that the former chromophore is more tightly held inside the bilayers. Furthermore, 2,6-MANS is found to possess the absorption and emission oscillators more closely oriented to the normal of membrane surface, while 1,8-ANS has both oscillators almost near the plane of the bilayers. The results furnish also a fair estimate of the random molecular motion own by the phospholipid molecules at room temperature. The comparison of the present data with those obtained from squid axon membranes confirms the validity of the proposed physical model, yielding a rough estimate of the axon membrane-area covered by integral protein macromolecules. These preliminary results derived from lecithin model membranes suggest that fluorescence polarization techniques can provide valuable informations if applied to study the macromolecular organization of in vitro reconstituted membranes.     

Orientation and rotational freedom of fluorescent probes in lecithin bilayers

Summary The fluorescence polarization properties of lecithin bilayers stained with 2,6-MANS and 1,8-ANS under applied potential steps have been studied. The fluorescence signal components of both dyes were found to have different sign and relative amplitude, suggesting that 1,8-ANS and 2,6-MANS behave differently when bound to black lipid membranes. In order to determine the location and the extent of rotational brownian motions of the bound chromophores, the experimental data were analyzed by using a simplified physico-mathematical model. According to it 2,6-MANS appears to have a ratio /gt higher than 1,8-ANS ( being the rotational relaxation of in plane rotations and the lifetime of the excited singlet state of the bound molecules), suggesting that the former chromophore is more tightly held inside the bilayers. Furthermore, 2,6-MANS is found to possess the absorption and emission oscillators more closely oriented to the normal of membrane surface, while 1,8-ANS has both oscillators almost near the plane of the bilayers. The results furnish also a fair estimate of the random molecular motion own by the phospholipid molecules at room temperature. The comparison of the present data with those obtained from squid axon membranes confirms the validity of the proposed physical model, yielding a rough estimate of the axon membrane-area covered by integral protein macromolecules. These preliminary results derived from lecithin model membranes suggest that fluorescence polarization techniques can provide valuable informations if applied to study the macromolecular organization of in vitro reconstituted membranes.Abbreviations 2,6-MANS 2-n-methylanilinonaphthalene-6-sulfonate - 1,8-ANS 1-anilinonaphthalene-8-sulfonate     

Alignment of anilinonaphthalene-sulfonate and related fluorescent probe molecules in squid axon membrane and in synthetic polymers

As a fluorescent probe for the squid axon membrane, the behavior of 1-anilinonaphthalene-8-sulfonate (1,8-ANS) was found to be very different from that of its positional isomer, 2,6-ANS, or of the methylated derivative, 2,6-TNS. The degree of polarization of the fluorescent light contributing to a transient intensity reduction during nerve excitation was larger than about 0.7 for both 2,6-ANS and 2,6-TNS, while the corresponding value for 1,8-ANS in a squid axon was about 0.35.The physicochemical basis of this difference was investigated by measuring the fluorescence polarization of these probe molecules incorporated into poly(vinyl alcohol) sheets. In a stretched sheet of this synthetic polymer, 1,8-ANS showed poor alignment, while the 2,6-derivatives were highly oriented with their transition moments aligned approximately in the direction of stretching. Based on these findings, the experimental results obtained from squid axons were interpreted as an indication of the existence, at or near the membrane, of a longitudinally oriented macromolecular structure, bringing about a high degree of alignment of 2,6-ANS or 2,6-TNS molecules.It is clear that, as a probe for fluorescence polarization studies of macromolecular structures, 2,6-TNS is far superior to 1,8-ANS.     

Fluorometry of turbid and absorbant samples and the membrane fluidity of intact erythrocytes.

In employing intrinsic or extrinsic fluorophores in the study of whole cells, or other strongly absorbant and/or scattering samples, the measured fluorescence intensity and polarization is seriously affected by absorption and scattering within the sample cuvet. These artifacts are analyzed and simple protocols are provided for overcoming them. An expression relating attenuation of the observed emission anisotropy to sample turbidity is derived. The validity of the method is confirmed by experiments in which the emission anisotropies and fluorescence yields of membrane probes in intact erythrocytes was measured with precision. It is also shown that the rotational mobility of the membrane probe 1-phenyl-3-(2-naphthyl)-2-pyrazoline is the same for intact erythrocytes and ghosts. These protocols are particularly useful in measuring the intrinsic fluorescence yield ratio for excimeric and monomeric emission of pyrene-containing membrane probes. This provides a method for determining the local lateral mobility of excimeric probes in intact erythrocytes.     

Fluorescence depolarization measurements on oriented membranes.

We describe the theory and experimental application of fluorescence depolarization measurements on small molecules bound to oriented phospholipid bilayers. The results yield insight into both the orientation and the rotational motion of fluorophores in a membrane environment. To accomplish this the angular distribution of polarized fluorescence intensities is measured on a membrane preparation consisting of stacked phospholipid bilayers oriented in a known coordinate system. Considerably more information is available from this data than in comparable solution phase measurements. Three parameters are derived from the data: the rate of rotational diffusion and the second and fourth degree order parameters. These latter two parameters provide an assessment of the average distribution of fluorophore orientation in the membrane bilayer. The data have been carefully examined for systematic experimental artifacts and new protocols are presented which help to eliminate errors that have not been amply treated in the past. We present data for two types of fluorescent molecules: (a) conventional membrane probes like diphenylhexatriene, perylene and anthroyloxy fatty acids; and (b) the anticancer agent adriamycin and several congeneric anthracycline antibiotics. The results show that the hydrocarbon core of membranes is more rigid than previously thought, particularly above the thermal phase transition temperature. We also show that the orientation of small molecules is sensitive to both the phospholipid composition and to the interaction of specific functional groups with the lipid bilayer. The results are discussed in terms of energetic models describing the general patterns for the binding of small molecules to biological membranes.     

Fluorescence Spectroscopy as a Tool to Unravel the Dynamics of Protein Nanoparticle Formation by Liquid Antisolvent Precipitation

Protein-based particles are very promising colloidal systems for protection and controlled release applications in the food, cosmetics and pharmaceutical sector. One technique to produce these protein colloidal particles is liquid antisolvent precipitation (LAS). Despite the simplicity and versatility of LAS, not much is known about the protein conformational changes and interactions that are at the basis of the particle formation process. In this study, steady state fluorescence experiments using intrinsic fluorophores were evaluated as a tool to unravel the dynamics of the protein nanoparticle formation. Colloidal whey protein isolate and gliadin particles were produced by LAS. Changes in particle diameter (distribution), polydispersity index and photophysical properties of intrinsic fluorophores were monitored as a function of antisolvent concentration. By combining dynamic light scattering with photophysical data, a model of the changes occurring during particle formation and disintegration could be proposed. The results suggest that particle formation and disintegration are fully reversible processes during which the main changes in protein conformation (around the fluorescent probes) occur at the same antisolvent concentrations. In principle, steady state fluorescence measurements using intrinsic probes can indeed be used to effectively report on (part of the) conformational changes for both protein systems under study.     

Simulation of structure, orientation, and energy transfer between AlexaFluor molecules attached to MscL

Measurements of time-resolved fluorescence anisotropy and fluorescence resonance energy transfer are finding many applications in the study of biological macromolecules as they enable structural properties of the host molecules to be determined in their natural environment. A difficulty in interpreting these experiments is that they both require knowledge of the relative orientation of the fluorophores, a property that is almost impossible to measure. Here we conduct simulations of AlexaFluor488 and AlexaFluor568 attached to two sites on the membrane channel MscL to provide an alternative mechanism for determining the likely configurations and orientational freedom of the fluorophores, as well as the most likely value of the orientation factor κ² for energy transfer between them. The fluorophores are relatively mobile, and are found to be more so when immersed in bulk water than when they interact with the lipid membrane. The fluorophores never insert deeply into the lipid, despite their hydrophobic linkers and aromatic headgroup structures. Properties such as the fluorescence anisotropy decay can be predicted from simulations of the fluorophores in bulk water that closely match experimental data. In contrast, when the fluorophores were attached to the large MscL protein it was difficult to sample all the possible configurations of the fluorophores due to the computational time required. While this approach is likely to provide useful data on solvent-accessible fluorophores attached to small proteins, simulations lasting >50 ns or the use of biasing forces are required to accurately predict orientation factors for use in energy transfer experiments on larger membrane-bound proteins.     

Effect of pulse electromagnetic radiation on erythrocyte ghosts

The pulse microwave radiation has been shown to increase the fluorescence intensity of 2-toluidinonaphthanene-6-sulfonate (2,6-TNS) and 1-anilinonaphthalene-8-sulfonate (1,8-ANS) built-in membranes of erythrocyte ghosts. In experiments with 2,6-TNS a frequency dependence of the effect of microwave radiation with maximum within the frequency range of 55-65 Hz has been found. It is suggested that the changes registered with fluorescent probes are induced by mechanical oscillations generated by the pulse microwave radiation.     

Ligand-induced thermostability in proteins: thermodynamic analysis of ANS-albumin interaction

A comparative thermodynamic study of the interaction of anilinonaphthalene sulfonate (ANS) derivatives with bovine serum albumin (BSA) was performed by using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The chemically related ligands, 1,8-ANS and 2,6-ANS, present a similar affinity for BSA with different binding energetics. The analysis of the binding driving forces suggests that not only hydrophobic effect but also electrostatic interactions are relevant, even though they have been extensively used as probes for non-polar domains in proteins. Ligand association leads to an increase in protein thermostability, indicating that both dyes interact mainly with native BSA. ITC data show that 1,8-ANS and 2,6-ANS have a moderate affinity for BSA, with an association constant of around 1-9x10(5) M(-1) for the high-affinity site. Ligand binding is disfavoured by conformational entropy. The theoretical model used to simulate DSC data satisfactorily reproduces experimental thermograms, validating this approach as one which provides new insights into the interaction between one or more ligands with a protein. By comparison with 1,8-ANS, 2,6-ANS appears as a more "inert" probe to assess processes which involve conformational changes in proteins.     

Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin

Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.     

Fluorescent lipid probes in the study of viral membrane fusion

Fluorescent lipid probes are widely used in the observation of viral membrane fusion, providing a sensitive method to study fusion mechanism(s). Due to the wealth of data concerning liposome fusion, a variety of fusion assays has been designed including fluorescent probe redistribution, fluorescence dequenching, fluorescence resonance energy transfer and photosensitized labeling. These methods can be tailored for different virus fusion assays. For instance, virions can be loaded with membrane dye which dequenches at the moment of membrane merger. This allows for continuous observation of fusion and therefore kinetic information can be acquired. In the case of cells expressing viral envelope proteins, dye redistribution studies of lipidic and water-soluble fluorophores yield information about fusion intermediates. Lipid probes can be metabolically incorporated into cell membranes, allowing observation of membrane fusion in vitro with minimal chance of flip flop, non-specific transfer and formation of microcrystals. Fluorescent lipid probes have been incorporated into liposomes and/or reconstituted viral envelopes, which provide a well-defined membrane environment for fusion to occur. Interactions of the viral fusion machinery with the membrane can be observed through the photosensitized labeling of the interacting segments of envelope proteins with a hydrophobic probe. Thus, fluorescent lipid probes provide a broad repertoire of fusion assays and powerful tools to produce precise, quantitative data in real time required for the elucidation of the complex process of viral fusion.     

Intrinsic fluorescence from DNA can be enhanced by metallic particles

High sensitivity detection of DNA is essential for genomics. The intrinsic fluorescence from DNA is very weak and almost all methods for detecting DNA rely on the use of extrinsic fluorescent probes. We show that the intrinsic emission from DNA can be enhanced many-fold by spatial proximity to silver island films. Silver islands are subwavelength size patches of metallic silver on an inert substrate. Time-resolved measurements show a decreased lifetime for the intrinsic DNA emission near the silver islands. These results of increased intensity and decreased lifetime indicate a metal-induced increase in the radiative rate decay of the DNA bases. The possibility of increased radiative decay rates for DNA bases and other fluorophores suggest a wide variety of DNA measurements and other biomedical assays based on metal-induced increases in the fluorescence quantum yield of weakly fluorescent substances.     

Determination of the orientational distribution and orientation factor for transfer between membrane-bound fluorophores using a confocal microscope

Orientational fluorophores have been a useful tool in physical chemistry, biochemistry, and more recently structural biology due to the polarized nature of the light they emit and that fact that energy can be transferred between them. We present a practical scheme in which measurements of the intensity of emitted fluorescence can be used to determine limits on the mean and distribution of orientation of the absorption transition moment of membrane-bound fluorophores. We demonstrate how information about the orientation of fluorophores can be used to calculate the orientation factor kappa(2) required for use in FRET spectroscopy. We illustrate the method using images of AlexaFluor probes bound to MscL mechanosensitive transmembrane channel proteins in spherical liposomes.     

Effects of tissue absorbance on NAD(P)H and Indo-1 fluorescence from perfused rabbit hearts

The effects of tissue optical absorbance on intracellular NAD(P)H and Indo-1 fluorescence emission have been evaluated in the perfused rabbit heart. These results demonstrate that the tissue optical absorbance significantly modifies the emission characteristics of these fluorophores. This tissue 'inner filter' effect, observed with both probes, changed as a function of tissue oxygenation and redox state in a wavelength-dependent manner. Pathlength calculations from these results indicate that this inner filter effect could occur with a mean pathlength of 310 microns due to the extremely high extinction coefficient of heart tissue. It is concluded that tissue optical absorbance significantly affects the fluorescent emission characteristics of both intrinsic and extrinsic probes in the intact heart, under a variety of conditions. Several potential methods of correcting for these tissue inner filter effects are discussed.     

An extended series of fluorescent sulfhydryl reagents useful in energy transfer measurements

Synthesis of four new fluorescent sulfhydryl reagents is described. All are isomers of the previously synthesized N-(iodoacetylaminoethyl)-1-naphthylamine-5-sulfonic acid (1,5-I-AEDANS) and its 1,8-isomer (1,8-I-AEDANS). Three of these new probes (1,4-Br-AEDANS, 2,8-Br-AEDANS, and 2,6-I-AEDANS) carry a single sulfonic acid residue and the fourth (3-(2,7)-Br-AEDANS) carries two sulfonic acid residues. The excitation and emission spectrum of each of these probes is distinct when covalently attached to bovine serum albumin. In addition, they all show a single fluorescent lifetime in the range of 8.0 to 20.8 nsec. This extended range of fluorescent isomers can thus be useful for selecting approprlate energy donors in fluorescence energy transfer experiments.     

Fluorescence studies on the interaction of myoglobin with mitochondria

To determine the nature and characteristic parameters of the myoglobin-mitochondrion interaction during oxymyoglobin (MbO₂) deoxygenation in the cell, we studied the quenching of the intrinsic mitochondrial flavin and tryptophan fluorescence by different liganded myoglobins in the pH range of 6–8, as well as the quenching of the fluorescence of the membrane probes 1,8-ANS and merocyanine 540 (M 540) embedded into the mitochondrial membrane. Physiologically active MbO₂ and oxidized metmyoglobin (metMb), which are unable to bind oxygen, were used as the quenchers. The absence of quenching of flavin and tryptophan fluorescence implies that myoglobin does not form quenching complexes with either electron transport chain proteins of the inner mitochondrial membrane or with outer membrane proteins. We found, however, that MbO₂ and metMb effectively quench 1,8-ANS and M 540 fluorescence in the pH range of 6–8. Characteristic parameters of 1,8-ANS and M 540 fluorescence quenching by the myoglobins (extent of quenching and quencher binding constant, K _m) are very similar, indicating that both probes are localized in phospholipid sites of the mitochondrial membrane, and myoglobin is complexed with these sites. The dependence of K _m on ionic strength proves the important role of coulombic interactions in the formation of the quenching complex. Since the overall charge of myoglobin is shown not to influence the K _m values, the ionic strength dependence must be due to local electrostatic interactions in which polar groups of some part of the myoglobin molecule participate. The most likely candidates to interact with anionic groups of mitochondrial phospholipids are invariant lysine and arginine residues in the environment of the myoglobin heme cavity, which do not change their ionization state in the pH range investigated.     

Single-molecule fluorescence resonance energy transfer techniques on rotary ATP synthases

Conformational changes of proteins can be monitored in real time by fluorescence resonance energy transfer (FRET). Two different fluorophores have to be attached to those protein domains which move during function. Distance fluctuations between the fluorophores are measured by relative fluorescence intensity changes or fluorescence lifetime changes. The rotary mechanics of the two motors of F(o)F(1)-ATP synthase have been studied in vitro by single-molecule FRET. The results are summarized and perspectives for other transport ATPases are discussed.     

Fluorescence as a method to reveal structures and membrane-interactions of amyloidogenic proteins

Amyloidogenesis is a characteristic feature of the 40 or so known protein deposition diseases, and accumulating evidence strongly suggests that self-association of misfolded proteins into either fibrils, protofibrils, or soluble oligomeric species is cytotoxic. The most likely mechanism for toxicity is through perturbation of membrane structure, leading to increased membrane permeability and eventual cell death. There have been a rather limited number of investigations of the interactions of amyloidogenic polypeptides and their aggregated states with membranes; these are briefly reviewed here. Amyloidogenic proteins discussed include A-beta from Alzheimer's disease, the prion protein, α-synuclein from Parkinson's disease, transthyretin (FAP, SSA amyloidosis), immunoglobulin light chains (primary (AL) amyloidosis), serum amyloid A (secondary (AA) amyloidosis), amylin or IAPP (Type 2 diabetes) and apolipoproteins. This review highlights the significant role played by fluorescence techniques in unraveling the nature of amyloid fibrils and their interactions and effects on membranes. Fluorescence spectroscopy is a valuable and versatile method for studying the complex mechanisms of protein aggregation, amyloid fibril formation and the interactions of amyloidogenic proteins with membranes. Commonly used fluorescent techniques include intrinsic and extrinsic fluorophores, fluorescent probes incorporated in the membrane, steady-state and lifetime measurements of fluorescence emission, fluorescence correlation spectroscopy, fluorescence anisotropy and polarization, fluorescence resonance energy transfer (FRET), fluorescence quenching, and fluorescence microscopy.