Protein structure probed by polarization spectroscopy. I. Evidence for fibrinogen rigidity from stationary fluorescence

Two fluorescent derivatives of human fibrinogen have been synthesized, by the covalent bonding of 1-dimethylaminoaphthalene-5-sulphonyl and methylpyrene chromophores, to investigate the internal molecular dynamics of the protein in solution. The stationary fluorescence depolarization of these derivatives under isothermal conditions is described here while in an accompanying paper (part II) a time-resolved study is reported. From the static fluorescence data it is concluded that reorientational processes in the subnanosecond and microsecond time ranges account for all the observed depolarization. The faster motion was assigned to the restricted, localized oscillations of the label while the slow motion was ascribed to the overall rotation of the protein molecule. Consequently, the protein in solution appears considerably rigid in the 10-1000 ns range, in contrast with a previous conception of a flexible fibrinogen based on non-isothermal depolarization experiments. These previous experiments are, in fact, concordant with the rigid fibrinogen proposed here if they are reinterpreted using Weber's early ideas on thermally activated depolarization (G. Weber, J. Biochem. 51 (1952) 145).     

Thermal unfolding process of dihydrolipoamide dehydrogenase studied by fluorescence spectroscopy

The thermal unfolding pathway for dihydrolipoamide dehydrogenase (LipDH) isolated from Bacillus stearothermophilus was investigated focusing on the transient intermediate state characterized through time-resolved fluorescence studies. The decrease in ellipticity in the far UV region in the CD spectrum, the fluorescence spectral change of Trp-91 and FAD, and the thermal enzymatic inactivation curve consistently demonstrated that LipDH unfolded irreversibly on heat treatment at higher than 65 degrees C. LipDH took a transient intermediate state during the thermal unfolding process which could refold back into the native state. In this state, the internal rotation of FAD was activated in the polypeptide cage and correspondingly LipDH showed a peculiar conformation. The transient intermediate state of LipDH characterized in time-resolved fluorescence depolarization studies showed very similar properties to the molten-globule state, which has been confirmed in many studies on protein folding.     

Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions. A new approximate solution of the rotational diffusion equation

We discussed the time-dependence of fluorescent emission anisotropy of a cylindrical probe in membrane vesicles. We showed that, if the motion of the probe were described as diffusion in an anisotropic environment, it would be possible to determine not only the second-rank but also the fourth-rank orientational order parameter from the decay of the fluorescence anisotropy. The approximations involved were based on an interpolation of short-time and long-time behavior of the relevant correlation functions. A general expression was derived for the time dependence of the fluorescence anisotropy in closed form, which applies to any particular distribution model. It was shown to be in good agreement with previously reported results for the cone model and the Gaussian model. Finally, the applicability of the theory to time-resolved and differential phase fluorescence depolarization experiments was discussed.     

Fluorescence depolarization dynamics of ionic strength sensors using time-resolved anisotropy

Eukaryotic cells exploit dynamic and compartmentalized ionic strength to impact a myriad of biological functions such as enzyme activities, protein-protein interactions, and catalytic functions. Herein, we investigated the fluorescence depolarization dynamics of recently developed ionic strength biosensors (mCerulean3-linker-mCitrine) in Hofmeister salt (KCl, NaCl, NaI, and Na₂SO₄) solutions. The mCerulean3-mCitrine acts as a Förster resonance energy transfer (FRET) pair, tethered together by two oppositely charged α-helices in the linker region. We developed a time-resolved fluorescence depolarization anisotropy approach for FRET analyses, in which the donor (mCerulean3) is excited by 425-nm laser pulses, followed by fluorescence depolarization analysis of the acceptor (mCitrine) in KE (lysine-glutamate), arginine-aspartate, and arginine-glutamate ionic strength sensors with variable amino acid sequences. Similar experiments were carried out on the cleaved sensors as well as an E6G2 construct, which has neutral α-helices in the linker region, as a control. Our results show distinct dynamics of the intact and cleaved sensors. Importantly, the FRET efficiency decreases and the donor-acceptor distance increases as the environmental ionic strength increases. Our chemical equilibrium analyses of the collapsed-to-stretched conformational state transition of KE reveal that the corresponding equilibrium constant and standard Gibbs free energy changes are ionic strength dependent. We also tested the existing theoretical models for FRET analyses using steady-state anisotropy, which reveal that the angle between the dipole moments of the donor and acceptor in the KE sensor are sensitive to the ionic strength. These results help establish the time-resolved depolarization dynamics of these genetically encoded donor-acceptor pairs as a quantitative means for FRET analysis, which complement traditional methods such as time-resolved fluorescence for future in vivo studies.     

Light-induced changes in the structure and accessibility of the cytoplasmic loops of rhodopsin in the activated MII state

Bovine rhodopsin was specifically labeled on the cytoplasmic surface at cysteine 140 (the first residue of the loop connecting helices III and IV) or at cysteine 316 (in the loop connecting helix VII and the palmitoylation sites) with the fluorescent labels fluorescein and Texas Red. These loops are involved in activation and signal transduction. The time-resolved fluorescence depolarization was measured in the dark state and in the M(II) state, with labeled samples consisting of rhodopsin-octylglucoside micelles or rod outer segment (ROS) membranes. In this way the diffusional dynamics of the flexible loops of rhodopsin were measured for the first time directly on the nanosecond time scale. Control experiments showed that the large number of weak excitation pulses required in these single photon counting experiments leads to <5% bleaching of the sample. Rhodopsin was trapped in the activated M(II) state for the duration of the fluorescence experiments ( approximately 20 min) after illumination at pH 6 and 5 degrees C. For both types of samples and at both labeled positions the dynamics of the label and loop motion as monitored by the time constants of the depolarization were not significantly different in the two states of the receptor. The end-anisotropy increased, however, from 0.09 in the dark to 0.16 in the M(II) state for ROS samples labeled at C140. The corresponding numbers for the C316 position are 0.06 and 0.12. Light-induced activation in M(II) is thus associated with a large increase in the loop steric hindrance due to a changed loop domain structure on the cytoplasmic surface. These results are supported by fluorescence quenching experiments with I(-), which indicate a significant decrease in the collisional quenching constant k(q) and in accessibility in the M(II) state at both positions. The rotational correlation time of the rhodopsin micelles increased from 48 ns in the dark state to 60 ns in M(II). This increase is caused by a change in volume and/or shape and is consistent with a structural change. These results demonstrate that time-resolved fluorescence depolarization is a powerful tool to study the changes in conformation and dynamics of the cytoplasmic loops that accompany the activation of rhodopsin and other G-protein coupled receptors.     

Construction and evaluation of a frequency-domain epifluorescence microscope for lifetime and anisotropy decay measurements in subcellular domains

The measurement of time-resolved fluorescence parameters in living cells provides a powerful approach to study cell structure and dynamics. An epifluorescence microscope was constructed to resolve multi-component fluorescence lifetimes and complex anisotropy decay rapidly in labile biological samples. The excitation source consisted of focused, polarized laser light modulated by an impulse-driven Pockels' cell; parallel acquisition of phase angles and modulation amplitudes at more than 40 frequencies (5-250 MHz) was obtained by multi-harmonic cross-correlation detection. Lifetime decay was measured against standard solutions introduced into the light path proximal to the microscope objective. Anisotropy decay was measured by rotation of a Glan-Thompson polarizer in the emission path. Phase reference light was split from the beam proximal to the microscope. Optical components were selected to avoid depolarization and to optimize fluorescence detection efficiency. The dichoric was replaced by a 1 mm square mirror. Fitting routine statistics were optimized for model discrimination in realistic biological samples. Instrument performance was evaluated using fluorescein in H2O/glycerol and H2O/ethylene glycol mixtures and in Swiss 3T3 fibroblasts in monolayer culture. Objective depolarization effects were evaluated by measurement of anisotropy decay using objectives of different numerical aperture. Lifetime and anisotropy decay measured by microscopy (0.5 micron laser spot) agreed with data obtained by cuvette fluorimetry. New biological applications for time-resolved fluorescence microscopy are discussed.     

Time-resolved single tryptophan fluorescence in photoactive yellow protein monitors changes in the chromophore structure during the photocycle via energy transfer

We show from time-resolved fluorescence intensity and depolarization experiments that the fluorescence of the unique tryptophan W119 of PYP is quenched by energy transfer to the 4-hydroxycinnamoyl chromophore. Whereas the intensity decay has a time constant of 0.18 ns in P, the decay in the absence of the cofactor (apo-PYP) has a single exponential lifetime of 4.8 ns. This difference in lifetime with and without acceptor can be explained quantitatively on the basis of energy transfer and the high-resolution X-ray structure of P, which allows an accurate calculation of the kappa2 factor. Fluorescence depolarization experiments with donor and acceptor indicate that both are immobilized so that kappa2 is constant on the fluorescence time scale. Using background illumination from an LED emitting at 470 nm, we measured the time-resolved fluorescence in a photostationary mixture of P and the intermediates I2 and I2'. The composition of the photostationary mixture depends on pH and changes from mainly I2 at low pH to predominantly I2' at high pH. The I2/I2' equilibrium is pH-dependent with a pKa of approximately 6.3. In I2 the lifetime increases to approximately 0.82 ns. This is not due to a change in distance or to the increase in spectral overlap but is primarily a consequence of a large decrease in kappa2. Kappa2 was calculated from the available X-ray structures and decreases from approximately 2.7 in P to 0.27 in I2. This change in kappa2 is caused by the isomerization of the acceptor, which leads to a reorientation of its transition dipole moment. We have here a rare case of the kappa2 factor dominating the change in energy transfer. The fluorescence decay in the light is pH-dependent. From an SVD analysis of the light/dark difference intensity decay at a number of pH values, we identify three species with associated lifetimes: P (0.18 ns), I2 (0.82 ns), and X (0.04 ns). On the basis of the pH dependence of the amplitudes associated with I2 and X, with a pKa of approximately 6.3, we assign the third species to the signaling state I2'. The absorption spectra of the 0.82 and 0.04 ns species were calculated from the pH dependence of their fluorescence amplitudes and of the photostationary light/dark difference absorption spectra. The lambda(max) values of these spectra (372 and 352 nm) identify the 0.82 and 0.04 ns components with I2 and I2', respectively, and validate the fluorescence data analysis. The mutant E46Q allows a further test of the energy transfer explanation, since lowering the pH in the dark leads to a bleached state with an increased spectral overlap but without the isomerization-induced decrease in kappa2. The measured lifetime of 0.04 ns is in excellent agreement with predictions based on energy transfer and the X-ray structure.     

Orientational properties of biological pigments in ordered systems studied with polarized light: photosynthetic pigment-protein complexes in membranes

A discussion is presented of the problems involved in the interpretation of linear dichroism and fluorescence depolarization experiments on macroscopically ordered membrane systems. Particular attention has been paid to ordered membranes containing photosynthetic pigment-protein complexes, but the mathematical treatment can equally well be applied to other systems. The information about the orientational properties of the pigments is obtained by the application of the theories developed for the characterization of the molecular orientational order in liquid-crystalline materials. It is shown that while linear dichroism only yields the order parameter S mu of the absorption transition moment, fluorescence depolarization experiments yield in addition the order parameter Sv of the emission transition moment as well as three orientational correlation functions of the two transition moments. It is argued that in general the latter information can only be obtained on utilizing a number of experimental scattering geometries. In particular, the merits of angle-resolved experiments are illustrated.     

Measurement of rotational dynamics by the simultaneous nonlinear analysis of optical and EPR data.

In the preceding companion article in this issue, an optical dye and a nitroxide radical were combined in a new dual function probe, 5-SLE. In this report, it is demonstrated that time-resolved optical anisotropy and electron paramagnetic resonance (EPR) data can be combined in a single analysis to measure rotational dynamics. Rigid-limit and rotational diffusion models for simulating nitroxide EPR data have been incorporated into a general non-linear least-squares procedure based on the Marquardt-Levenberg algorithm. Simultaneous fits to simulated time-resolved fluorescence anisotropy and linear EPR data, together with simultaneous fits to experimental time-resolved phosphorescence anisotropy decays and saturation transfer EPR (ST-EPR) spectra of 5-SLE noncovalently bound to bovine serum albumin (BSA) have been performed. These results demonstrate that data from optical and EPR experiments can be combined and globally fit to a single dynamic model.     

Fluorescence lifetimes: fundamentals and interpretations

Fluorescence measurements have been an established mainstay of photosynthesis experiments for many decades. Because in the photosynthesis literature the basics of excited states and their fates are not usually described, we have presented here an easily understandable text for biology students in the style of a chapter in a text book. In this review we give an educational overview of fundamental physical principles of fluorescence, with emphasis on the temporal response of emission. Escape from the excited state of a molecule is a dynamic event, and the fluorescence emission is in direct kinetic competition with several other pathways of de-excitation. It is essentially through a kinetic competition between all the pathways of de-excitation that we gain information about the fluorescent sample on the molecular scale. A simple probability allegory is presented that illustrates the basic ideas that are important for understanding and interpreting most fluorescence experiments. We also briefly point out challenges that confront the experimenter when interpreting time-resolved fluorescence responses.     

A new analysis method for the membrane viscosity from steady-state fluorescence depolarization

The absolute value of the viscosity in membrane lipid bilayers, which is different from the microviscosity advocated by Shinitzky, could be calculated from steady-state fluorescence depolarization of a hydrocarbon fluorophore, 1,6-diphenyl-1,3,5-hexatriene (DPH). This method was based on the theory of time-resolved fluorescence anisotropy and empirical relationships between fluorescence life time and the anisotropy parameters such as half cone angle in wobbling motion and wobbling diffusion rate of the fluorescent probe. Obtained viscosity values of various membranes from this method were consistent with those from time resolved method within experimental error.     

Self-aggregation--an intrinsic property of G(M1) in lipid bilayers

We demonstrate that the ganglioside G(M1) in lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exhibits a non-uniform lateral distribution, i.e., enriched regions of GM(1) molecules are formed, which is an argument in favour of self-aggregation of G(M1) being an intrinsic property of G(M1) ganglioside. This was concluded from energy transfer/migration studies of BODIPY-labelled gangliosides by means of time-resolved fluorescence lifetime and depolarization experiments. Three fluorophore-labelled gangliosides were synthesized to include either of two spectroscopically different BODIPY groups. These were specifically localized either in the polar headgroup region or in the non-polar region of the lipid bilayer. An eventual ganglioside-ganglioside affinity/aggregation induced by the BODIPY groups was experimentally excluded, which suggests their use in examining the influence of G(M1) in more complex systems.     

Protein structure probed by polarization spectroscopy. II. A time-resolved fluorescence study of human fibrinogen

Human fibrinogen in solution was studied by monitoring the time-resolved depolarization of the fluorescence emitted by two spectroscopic labels of which the fluorescence lifetimes differ by an order of magnitude. Contrary to a long-held view, no evidence of molecular flexibility was found in the 10-1000 ns range. In addition, from the rate of the overall rotation, it is proposed that a prolate and symmetric ellipsoid of 47 X 10.5 nm may represent the time-averaged hydrodynamic size and shape of the protein in solution. This rigid and highly hydrated structure (4 g water/g protein) accommodates the latest nodular models obtained from electron microscopy, explains the singular hydrodynamics of fibrinogen and, apparently, it would perform the two main functions of the protein in haemostasis, blood coagulation and platelet aggregation, more efficiently than the flexible molecule.     

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.     

The partially unfolded state of beta-momorcharin characterized with steady-state and time-resolved fluorescence studies

The specific conformation of partially unfolded state of beta-momorcharin was characterized through the steady-state and time-resolved fluorescence spectroscopic studies on a single Trp-190 which located adjacently to the active site. The content of secondary structure was retained, the binding of ANS was remarkably enhanced, and the correlation time of entire protein rotation was prolonged at the partially unfolded state formed by being equilibrated with the mild concentration of guanidine hydrochloride. The time-resolved fluorescence depolarization and excitation energy transfer analysis suggest that Trp-190 approached 2 A closer to Tyr-70 and was hidden from the exposure to the protein surface, while the rotational correlation time and freedom of its segmental motion were shortened and enhanced, respectively. These results suggest that the transient folding/unfolding intermediate state of beta-momorcharin adopt the specific conformation at the vicinity of the active site, although it exhibits very similar properties with those of the generally known molten-globule state.     

Homo-FRET Imaging Enables Quantification of Protein Cluster Sizes with Subcellular Resolution

Fluorescence-anisotropy-based homo-FRET detection methods can be employed to study clustering of identical proteins in cells. Here, the potential of fluorescence anisotropy microscopy for the quantitative imaging of protein clusters with subcellular resolution is investigated. Steady-state and time-resolved anisotropy detection and both one- and two-photon excitation methods are compared. The methods are evaluated on cells expressing green fluorescent protein (GFP) constructs that contain one or two FK506-binding proteins. This makes it possible to control dimerization and oligomerization of the constructs and yields the experimental relation between anisotropy and cluster size. The results show that, independent of the experimental method, the commonly made assumption of complete depolarization after a single energy transfer step is not valid here. This is due to a nonrandom relative orientation of the fluorescent proteins. Our experiments show that this relative orientation is restricted by interactions between the GFP barrels. We describe how the experimental relation between anisotropy and cluster size can be employed in quantitative cluster size imaging experiments of other GFP fusions. Experiments on glycosylphosphatidylinisotol (GPI)-anchored proteins reveal that GPI forms clusters with an average size of more than two subunits. For epidermal growth factor receptor (EGFR), we observe that ∼40% of the unstimulated receptors are present in the plasma membrane as preexisting dimers. Both examples reveal subcellular heterogeneities in cluster size and distribution.     

13C and 15N NMR and time-resolved fluorescence depolarization study of bovine carbonic anhydrase--4-methylbenzenesulfonamide complex

The influence of the binding of the high-affinity inhibitor, 4-methylbenzenesulfonamide, to the active site of bovine carbonic anhydrase B was studied by 15N- and 13C-NMR spectroscopy. The rotational correlation time dependence on temperature and concentration of the complex was determined by time-resolved fluorescence depolarization measurements. Our experiment provides evidence that the stoichiometry of the interaction of 4-methylbenzenesulfonamide with carbonic anhydrase B is 1:1 and the inhibitor is bound in anionic form. The 15N-NMR relaxation parameters confirm our previous conclusions about the presence of librational motions in the active site of carbonic anhydrase and indicate that the internal motion in the enzyme-inhibitor complex is more restricted than the backbone motion in the uncomplexed native enzyme.     

Mechanism of acridine orange interaction with phospholipids and proteins in renal microvillus vesicles

The mechanism of interaction of acridine orange (AO), a fluorescent, weak base, with rabbit kidney brush border membrane vesicles (BBMV) has been studied by absorption, and steady-state and time-resolved fluorescence spectroscopy. Equilibrium binding experiments indicate that AO binds to an apparent single class of sites on BBMV with a dissociation constant of 90 microM and site stoichiometry of 810 nmol/mg protein. The absorption spectra AO indicate that BBMV induces aggregation of AO; experiments with lipid vesicles show that the aggregation requires BBMV membrane proteins. Fluorescence stopped-flow experiments in which 0.15 mg/ml BBMV is mixed with increasing concentrations of AO result in a time course of fluorescence enhancement for [AO] less than 1.5 microM, and of fluorescence quenching for [AO] greater than 1.5 microM. Similar stopped-flow experiments with phosphatidylcholine lipid vesicles result only in a fluorescence enhancement time course. These results indicate the presence of two parallel pathways for AO binding to BBMV: one for AO binding to BBMV lipid, the other for AO binding to BBMV protein. Nanosecond lifetime measurements and fluorescence titration experiments confirm the presence of two environments for AO in BBMV. Fluorescence stopped-flow experiments indicate that AO responds to the imposition of an outwardly directed proton gradient by a rapid (less than 0.5 s) decrease in fluorescence, corresponding to re-equilibration of AO into the acidic intravesicular compartment, followed by an increase in fluorescence, corresponding to proton flux across the membrane. These findings have been incorporated into a stepwise mechanism for AO interaction with BBMV which have direct implications for the use of AO as a pH indicator in biological systems.     

Binding of camphor to Pseudomonas putida cytochrome p450(cam): steady-state and picosecond time-resolved fluorescence studies

The binding of camphor to cytochrome P450(cam) has been investigated by steady-state and time-resolved tryptophan fluorescence spectroscopy to obtain information on the substrate access channel. The fluorescence quenching experiments show that some of the tryptophan residues undergo changes in their local environment on camphor binding. The time-resolved fluorescence decay profile gives four lifetime components in the range from 99 ps to 4.5 ns. The shortest lifetime component assigned to W42 lies close to the proposed camphor access channel. The results show that the fluorescence of W42 is greatly affected on binding of camphor, and supports dynamic fluctuations involved in the passage of camphor through the access channel as proposed earlier on the basis of crystallographic, molecular dynamics simulation and site-directed mutagenesis studies.