Fluorescence properties of labeled proteins near silver colloid surfaces

The fluorescence properties of a monolayer of labeled avidin molecules were studied near silver island films. We first adsorbed a monolayer of biotinylated-BSA as a base that was used to capture labeled avidin molecules. For labeled avidin on silver island films, we observed an increase of the fluorescence intensity of between 18 and 80 with one-photon excitation and up to several hundredfold or larger with two-photon excitation. The probes were moderately more photostable in the presence of silver islands. There was also a dramatic decrease in the lifetimes with the amplitude-weighted values decreasing from 7- to 35-fold. The data suggest that these spectral changes are due to both increased rates of excitation near the metallic particles and increases in the rates of radiative decay. Because these silver island surfaces are very heterogeneous, we are hopeful that larger increases in intensity and photostability can be obtained for probes situated at an optimal distance from the ideal island surfaces.     

Fluorescence study of the thyroxine-dependent conformational changes in human serum transthyretin.

Fluorescence studies of transthyretin (TTR) were conducted to detect structural changes associated with the environment of its two tryptophans, induced by binding of thyroxine (T4). Non-radiative tryptophans relaxation rate has an activation energy of 6.4 kcal/mol for TTR, which is decreased to 4.4 kcal/mol for TTR-T4 complex. The maximum fluorescence wavelength was red-shifted as the excitation wavelength was increased. T4 changed the magnitude of this shift. T4 binding per se changed the emission maximum reflecting different environments of the tryptophans. Double-quenching experiments also showed that T4 produces changes in the tryptophans environments. These findings were interpreted as the result of structural alterations in the protein matrix induced by T4 which contribute in part to explain the negative cooperativity associated with the occupancy of the second binding site.     

Fluorescence spectral properties of labeled thiolated oligonucleotides bound to silver particles

We examined the fluorescent spectral properties of fluorescein-labeled DNA oligomers when directly bound to metallic silver particles via a terminal sulfhydryl group. We found a 12-fold increase in fluorescence intensity and 25-fold decrease in lifetime for a fluorescein residue positioned 23 nucleotides from the silver surface compared to labeled oligomers in free solution. Similar results were found for a 23-mer labeled with five fluorescein residues. The absence of long lifetime components in the intensity decays suggests that all labeled oligomers are bound to silver and affected similarly by the metallic surfaces. These results provide the basic knowledge needed to begin use of metal-enhanced fluorescence for the detection of target sequences in simple formats potentially without a washing separation step. The use of metal-enhanced fluorescence provides a generic approach to obtaining a hybridization-dependent increase in fluorescence with most, if not all, commonly used fluorophores.     

Fluorescence of equine platelet tropomyosin labeled with acrylodan

Equine platelet tropomyosin was labeled with the sulfhydryl-specific fluorescent reagent 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). The extent of labeling at 4 degrees C could be regulated between 0.5 and 1.3 acrylodans per tropomyosin chain by varying the reaction time from 1 to 4.5 h. Acrylodan-labeled platelet tropomyosin, AD-P-TM, was highly fluorescent, having an emission maximum near 518 nm on excitation at 365 nm. Steady-state measurements of polarization of the fluorescence of AD-P-TM in both low and high ionic strength solutions gave Perrin plots that exhibited sharp changes in slope near 50 degrees C, indicative of a sharp increase in mobility of the label at that temperature. This correlates with the melting temperature of the platelet tropomyosin coiled coil observed by circular dichroism [G. P. C?té, W. G. Lewis, M. D. Pato, and L. B. Smillie, (1978) FEBS Lett. 91, 237-241]. Perrin plots of carboxypeptidase A-treated platelet tropomyosin that was labeled with acrylodan after digestion resembled more closely those of acrylodan-labeled cardiac tropomyosin rather than those of AD-P-TM, suggesting that the observed emission arose from label at Cys-153 on each truncated platelet tropomyosin chain. In solutions containing 150 mM KCl and 5 mM MgCl2, addition of actin at up to a sixfold molar excess over AD-P-TM caused both the fluorescence emission intensities and fluorescence polarization values of samples to increase. In the presence of actin, the wavelength of maximal emission was shifted to shorter values by about 5 to 7 nm. These changes indicate that actin does bind to AD-P-TM and that the binding affects the environment of the label, both by making it more hydrophobic and by reducing the freedom of the label to tumble in solution.     

Fluorescence properties and conformational stability of the beta-hemocyanin of Helix pomatia

The beta-hemocyanin (beta-HpH) is one of the three dioxygen-binding proteins found freely dissolved in the hemolymph of the gastropodan mollusc Helix pomatia. The didecameric molecule (molecular mass 9 MDa) is built up of only one type of subunits. The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Upon excitation of the hemocyanins at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-beta-HpH. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-form. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the beta-HpH and its subunits exist in different conformations. The thermal stability of the oxy- and apo-beta-HpH is characterized by a transition temperature (Tm) of 84 degrees C and 63 degrees C, respectively, obtained by differential scanning calorimetry. Increase of the temperature influences the active site at lower temperatures than the environments of tryptophans and tyrosines causing a loss of oxygen bound to the copper atoms. This process is, at least partially, reversible as after cooling of the protein samples, around 60% reinstatement of the copper-peroxide band has been observed. The results confirm the role of the copper-dioxygen complex for the stabilization of the hemocyanin structure in solution. The other important stabilizing factor is oligomerization of the hemocyanin molecule.     

Fluorescence photobleaching recovery in solutions of labeled actin.

We have demonstrated that the technique of fluorescence photobleaching recovery (FPR) can be used to examine the state of a single component in complex self-assembling macromolecular systems. Polymerization of actin, initiated by addition of salt or Mg+2 to a low-ionic-strength solution of G-actin, has been observed by sequential measurement of FPR with the aid of fluorescein-labeled actin. Solutions of actin which had been labeled using 5-iodoacetamido fluorescein (5-IAF) showed anomalous recovery of fluorescence above the initial value, which indicates a photoinduced increase in local polymerization. No such anomaly was observed with actin that had been labeled with fluorescein isothiocyanate (FITC). The FPR data are directly interpretable in terms of the fraction of labeled protein that is immobilized in the supramolecular assembly and in terms of the average diffusion coefficient of the mobile fraction. Our data are consistent with the "treadmill" model of actin polymerization, in that they show that actin is present under polymerizing conditions either as a high polymer or as monomer or low oligomer. We believe that the FPR technique can be applied to the study of many types of reconstituted motile or cytoskeletal systems in vitro or in vivo.     

Fluorescence study of conformational transitions in the structure of myoglobin

Fluorescence studies of myoglobin and Mb-like structures, apomyoglobin and the complex of apo-Mb with protoporphyrin IX, reveal both the similarity between them, which is due to a common type of polypeptide chain folding, and the distinctions imposed by the influence of the prosthetic group. Close resemblance of structures of holomyoglobin and its metal-free analog, PPIX--apo-Mb, points to a key role of specific interactions between the protein and the protoporphyrin macrocycle rather than the Fe-protein bond in the formation of Mb-like structures. In PPIX--apo-Mb, both the hydrophobic core and the important ionic bonds between different structural elements () stabilizing the Mb structure are almost completely retained. The bond between Fe and proximal His-F8 allows additional integration of the structures of the heme cavity and the myoglobin molecule as a whole, providing its functional activity and highly cooperative conformational transitions. In all the myoglobin-like structures studied, a certain relationship is found between conformational states of the , the heme cavity, and the N-terminal part of the molecule. This is probably due to variations in the mutual orientation of the ABCDE and FGH helical domains, depending on the interactions between the protein, the prosthetic group, and the ligand in the heme crevice. The correlation between conformations of the N-terminal and heme regions found at a level of the globin tertiary structure is very important for understanding the mechanisms of homo- and heterotropic regulation in tetrameric hemoglobins.     

Fluorescence spectral properties of cyanine dye labeled DNA near metallic silver particles

Recent studies have demonstrated that silver metallic particles can increase the quantum yield and decrease the lifetimes of nearby fluorophores. These studies are extended to double stranded DNA oligomers labeled with N,N'-(dipropyl)-tetramethylindocarbocyanine (Cy3) or N,N-(dipropyl)-tetramethylindodicarbocyanine (Cy5). The proximity to silver particles increases the apparent quantum yields and decreases the lifetimes of the double helical DNA 23-mer labeled individually with Cy3 or Cy5. The decreased lifetimes are accompanied by apparently increased photostability of the labeled oligomers near silver particles. Because of spatial averaging across the sample these results are likely to significantly underestimate the effects of silver particles on labeled DNA localized at an optimal distance from the metallic surface. These results suggest that DNA arrays fabricated on substrates with silver particles can display increased sensitivity and photostability in the analysis of gene expression.     

Fluorescence conformational probe study of calcium release from sarcoplasmic reticulum

Sarcoplasmic reticulum isolated from rabbit skeletal muscle was labeled with a limited (0.625 nmol/mg sarcoplasmic reticulum protein) amount of the fluorescent thiol reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM). The fluorescence intensity of the membrane-attached DACM decreased concurrently with (Ca2+ and caffeine)-induced Ca2+ release, depolarization-induced Ca2+ release and Ca2+-dependent dependent passive efflux of Ca2+. The decreased DACM fluorescence level initiated by a Ca2+ jump was subsequently reversed under passive efflux conditions when there was no ATP-dependent Ca2+ uptake, suggesting spontaneous closing of the channels. Therefore, the higher fluorescence level corresponds to a larger population of closed channels, whereas the lower level represents a larger population of opened channels. Under conditions when the Ca2+ release-coupled fluorescence change was maximal, a stoichiometric incorporation of DACM took place only into a 32-kDa protein. Furthermore, reconstituted vesicles, in which purified DACM-labeled 32-kDa protein was incorporated into unlabeled sarcoplasmic reticulum vesicles, were capable of both (Ca2+ and caffeine)-induced Ca2+ release and the release-coupled DACM fluorescence change. These results suggest that the 32-kDa protein is a constituent of the Ca2+ release channel or a protein which is in close contact with the channel.     

Transport properties of single-file pores with two conformational states.

Complex facilitative membrane transporters of specific ligands may operate via inner channels subject to conformational transitions. To describe some properties of these systems, we introduce here a kinetic model of coupled transport of two species, L and w, through a two-conformational pore. The basic assumptions of the model are: a) single-file of, at most, n molecules inside the channel; b) each pore state is open to one of the compartments only; c) there is at most only one vacancy per pore; d) inside the channel, a molecule of L occupies the same positions as a molecule of w; and e) there is at most only one molecule of L per pore. We develop a general representation of the kinetic diagram of the model that is formally similar to the one used to describe one-vacancy transport through a one-conformational single-file pore. In many cases of biological importance, L could be a hydrophilic (ionic or nonionic) ligand and w could be water. The model also finds application to describe solute (w) transport under saturation conditions. In this latter case, L would be another solute, or a tracer of w. We derive steady-state expressions for the fluxes of L and w, and for the permeability coefficients. The main results obtained from the analysis of the model are the following. 1) Under the condition of equilibrium of w, the expression derived for the flux of L is formally indistinguishable from the one obtainable from a standard four-state model of ligand transport mediated by a two-conformational transporter. 2) When L is a tracer of w, we can derive an expression for the ratio between the main isotope and tracer permeability coefficients (Pw/Pd). We find that the near-equilibrium permeability ratio satisfies (n - 1) < or = (Pw/Pd)eq < or = n, a result previously derived for the one-conformational, single-file pore for the case that n > or = 2. 3) The kinetic model studied here represents a generalization of the carrier concept. In fact, for the case that n = 1 (corresponding to the classical single-occupancy carrier), the near-equilibrium permeability ratio satisfies 0 < or = (Pw/Pd)eq < or = 1, which is characteristic of a carrier performing exchange-diffusion.     

Synthesis and properties of fluorescent labeled detergents analogues of glycocholic acid

For studying membrane processes with participation of detergents, fluorescent analogues of glycocholic acid containing p-hydroxybenzyl, 7-nitrobenz-2-oxa-1,3-diazol-4-yl, or fluorescein-5-thiocarbamoyl fluorophore in the glycyl moiety attached to glycocholic acid were synthesized. The fluorophores are in the probes near their carboxyl groups and, in membrane systems, should therefore be situated on the interface and be sensitive to phase transitions. The critical micelle concentrations were determined for the analogues and found to be close to those of cholate and glycocholate in the case of the first two compounds. We presume that the behavior of the probes in membrane systems will mimic the behavior of the bile acid salts.     

Studies on conformational changes in Na,K-ATPase labeled with 5-iodoacetamidofluorescein

The rates of individual steps in the reaction cycle of dog kidney Na,K-ATPase labeled with iodoacetamidofluorescein (IAF) were measured using the fluorescence stopped-flow technique. The maximal rate of the fluorescence quenching accompanying ATP hydrolysis at 20 degrees C in the presence of K+ is 66.3 s-1, while the turnover rate in the same conditions is 15.5 s-1. The rate without K+ is slightly lower. Unexpectedly, at very high ionic strength, K+ accelerates the rate 2-fold. The fluorescence change appears to be associated with the E1P----E2P transition. The results are consistent with the classical Albers-Post scheme but do not support recent criticisms that E1P is kinetically incompetent in the presence of Na+ plus K+. As expected, in the absence of ATP the rate of E2(K)----E1Na was very slow (0.2 s-1) but was greatly accelerated by ATP (maximal rate 15.9 s-1) with low affinity (K0.5 = 196 microM). It was concluded that E2(K)----E1 is the slowest step of the cycle, even at nonlimiting ATP concentrations. The rate of E1K----E2(K) for both IAF- and fluorescein 5'-isothiocyanate-labeled enzyme was stimulated by K+ acting with low affinity, but not at all by ATP at 5 microM. Whereas the maximal rate with IAF-enzyme (271 s-1) was similar to previous work, the K+ affinity was significantly higher. Fluorescence signals accompanying hydrolysis of acetyl phosphate with both IAF- and fluorescein 5'-isothiocyanate-labeled enzyme have similar rates, 5.25 s-1 and 4.06 s-1, respectively. A species difference was observed between dog and pig kidney Na,K-ATPase in that both enzymes are labeled with IAF but only in dog enzyme were conformational transitions associated with fluorescence changes. Therefore, the IAF-labeled dog kidney enzyme is the preparation of choice for measuring fluorescence changes accompanying ATP hydrolysis.     

Solution conformational study of Scyliorhinin I analogues with conformational constraints by two-dimensional NMR and theoretical conformational analysis.

Two analogues of Scyliorhinin I (Scyl), a tachykinin with N-MeLeu in position 8 and a 1,5-disubstituted tetrazole ring between positions 7 and 8, introduced in order to generate local conformational constraints, were synthesized using the solid-phase method. Conformational studies in water and DMSO-d6 were performed on these peptides using a combination of the two-dimensional NMR technique and theoretical conformational analysis. The algorithm of conformational search consisted of the following three stages: (i) extensive global conformational analysis in order to find all low-energy conformations; (ii) calculation of the NOE effects and vicinal coupling constants for each of the low energy conformations; (iii) determining the statistical weights of these conformations by means of a nonlinear least-squares procedure, in order to obtain the best fit of the averaged simulated spectrum to the experimental one. In both solvents the three-dimensional structure of the analogues studied can be interpreted only in terms of an ensemble of multiple conformations. For [MeLeu8]Scyl, the C-terminal 6-10 fragment adopts more rigid structure than the N-terminal one. In the case of the analogue with the tetrazole ring in DMSO-d6 the three-dimensional structure is characterized by two dominant conformers with similar geometry of their backbones. They superimpose especially well (RMSD = 0.28 A) in the 6-9 fragments. All conformers calculated in both solvents superimpose in their C-terminal fragments much better than those of the first analogue. The results obtained indicate that the introduction of the tetrazole ring into the Scyl molecule rigidifies its structure significantly more than that of MeLeu.     

Fluorescence properties of terbium-alkaline phosphatase.

The addition of Tb³⁺ to apoalkaline phosphatase at pH 8.0 results in the formation of a metalloprotein with an enhanced Tb³⁺ fluorescence at 492, 545, and 580 nm. The Tb³⁺ excitation spectrum is most consistent with a process in which energy is transferred from one or more tyrosyl chromophores to the bound lanthanide. An analysis of the fluorescence data under equilibrium conditions yields one Tb³⁺ binding site per enzyme dimer with a K_n = 0.16 ± 0.02 μm. The Tb³⁺-alkaline phosphatase complex is not catalytically active nor does it incorporate covalently bound phosphate, but the specific activity of Zn²⁺-alkaline phosphatase is significantly enhanced in the presence of Tb³⁺ indicating that this lanthanide mimics Mg²⁺ in stabilizing the structure of alkaline phosphatase. The fluorescence of the Tb³⁺-enzyme is found to be quite sensitive to conformational changes which occur upon addition of Zn²⁺ or substrates.     

Fluorescence resonance energy transfer in ferritin labeled with multiple fluorescent dyes

We simultaneously labeled ferritin with two Alexa Fluor fluorophores (AF350 and AF430). When both fluorophores label the same ferritin subunit, fluorescence resonance energy transfer (FRET) takes place from the excited AF350 to the acceptor AF430. By varying the number and the ratio of labeling fluorophores, we can modulate FRET such that the ferritin particles can exhibit multiple colors under UV illumination. Labeling of the ferritin shell does not affect the properties of the metallic core. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.