Proximity of lectin receptors on the cell surface measured by fluorescence energy transfer in a flow system.

Molecules of the lectin concanavalin A have been labeled separately with the fluorescein and rhodamine chromophores and jointly bound to the surface of transformed Friend erythroleukemia cells. The two dyes constitute an ideal donor-acceptor pair for fluorescence resonance energy transfer thereby permitting the determination of the proximity relationships between bound ligand molecules and the corresponding surface receptors. The transfer efficiency at saturation (about 57%) was measured in a multiparameter flow system using laser excitation at 488 nm and detection of fluorescein and rhodamine emission intensities as well as the emission anisotropy of the rhodamine fluorescence for each cell. The degree of energy transfer was estimated from the quenching of donor emission, the sensitization of acceptor emission, and the depolarization of acceptor fluorescence. The system has been modeled according to a formalism developed by Gennis and Cantor (Biochemistry 11: 2509, 1972). We estimate the separation between the surfaces of bound lectin molecules at saturation to be 0-40 A, a range possibly characteristic for micropatches induced by ligand binding.     

Laser-assisted fluorescence microscopy for measuring cell membrane dynamics.

Membranes of living cells are characterized by laser-assisted fluorescence microscopy, in particular a combination of microspectrofluorometry, total internal reflection fluorescence microscopy (TIRFM), fluorescence lifetime imaging (FLIM) and Forster resonance energy transfer (FRET) spectroscopy. The generalized polarization (GP, characterizing a spectral shift which depends on the phase of membrane lipids) as well as the effective fluorescence lifetime (tau(eff)) of the membrane marker laurdan were revealed to be appropriate parameters for membrane stiffness and fluidity. GP decreased with temperature, but increased during cell growth and was always higher for the plasma membrane than for intracellular membranes. Microdomains of different fluorescence lifetimes tau(eff) were observed at temperatures above 30 degree C and disappeared during cell aging. Non-radiative energy transfer was used to detect laurdan selectively in close proximity to a molecular acceptor (DiI) and may present a possibility for measuring membrane dynamics in specific microenvironments.     

Spatial relationship between the prodan site,Trp-214, and Cys-34 residues in human serum albumin and loss of structure through incremental unfolding

Prodan (6-propionyl-2-(dimethylamino)-naphthalene), a competitive inhibitor of warfarin binding to human serum albumin (HSA) at drug site I, was used to determine the inter- and intradomain distances of HSA. The fluorescence resonance energy transfer (FRET) distances between prodan and Trp-214, prodan and 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CM)-modified Cys-34, and Trp-214 and CM-Cys-34 were determined to be 25.5 +/- 0.5 A, 33.1 +/- 0.8 A, and 32.4 +/- 1 A, respectively. FRET analysis showed that low concentration of palmitic acid (5 microM) increased the interdomain distance between the Trp-214 in domain II and CM-Cys-34 in domain I by approximately 5 A without perturbing the secondary structure of HSA and the immediate environment of Trp-214. Palmitic acid (5 microM) increased the prodan fluorescence by increasing the quantum yield of bound prodan without altering the tryptophan environment. However, palmitic acid (>10 microM) decreased the prodan fluorescence and increased the tryptophan fluorescence. Our results indicate that the high affinity palmitic acid binding site is located at the interface of domains I and II. On the basis of our measurements, a schematic model representing the drug site-1, Trp-214, and Cys-34 along with the palmitic acid sites has been constructed. In addition, prodan fluorescence, FRET, and ligand binding were used to monitor guanidine hydrochloride-induced denaturation of HSA. An analysis of the equilibrium unfolding data suggests that HSA undergoes a two-state unfolding transition with no detectable intermediate. However, kinetic analysis using multiple probes and thermal denaturation studies showed that the unfolding of the prodan site in HSA preceded the unfolding of tryptophan environment. In addition, the separation of domain I and II occurred before the global unfolding of the protein. The data support the idea that HSA loses its structure incrementally during its unfolding.     

Bifluorophoric molecules as fluorescent beacons for antibody-antigen binding

The quenching of fluorescence (up to 98%) by anti-fluorescein antibodies is well documented in the literature. Here we report a system where, instead of quenching, bifluorophoric molecules are designed to increase in fluorescence upon binding by an anti-fluorescein antibody. Bifluorophoric molecules are made of fluorescein (F) linked to tetramethylrhodamine (T) via varying numbers of methylene units, denoted as F-(CH(2))(n)-T. These F-(CH(2))(n)-T conjugates are almost nonfluorescent when free in solution due to intramolecular dimerization and stacking. Upon binding to an anti-fluorescein antibody, however, up to 110-fold increase in fluorescence was observed from the rhodamine moiety. This increase is believed to result from intramolecular dimer dissociation that dequenches the rhodamine fluorescence. Fluorescein fluorescence, on the other hand, remains quenched due to binding and intramolecular resonance energy transfer. Moreover, the excitation wavelength was at the absorption maxima of fluorescein, giving a Stoke's shift of about 90 nm. This system couples directly molecular recognition with a concurrent increase in fluorescence emission, obviating wash and incubation steps required by most assays. It is an important molecular reporter system for developing homogeneous assays.     

Triacontanol and Jasmonic Acid Differentially Modulate the Lipid Organization as Evidenced by the Fluorescent Probe Behavior and <Superscript>31</Superscript>P Nuclear Magnetic Resonance Shifts in Model Membranes

Fluorescence resonance energy transfer (FRET), time-resolved fluorescence and anisotropy decays were determined in large unilamellar vesicles (LUVs) of egg phosphatidylcholine with the FRET pair N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dipalmitoyl-sn-glycero-3-phospho-ethanolamine as donor and lissamine rhodamine B 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine as acceptor, using 2-ps pulses from a Ti:sapphire laser on LUVs with incorporated plant growth regulators: triacontanol (TRIA) and jasmonic acid (JA). FRET efficiency, energy transfer rate, rotation correlation time, microviscosity, and diffusion coefficient of lateral diffusion of lipids were calculated from these results. It was observed that TRIA and JA differentially modulated all parameters studied. The effect of JA in such modulations was always partially reversed by TRIA. Also, the generalized polarization of laurdan fluorescence indicated that JA enhances the degree of hydration in lipid bilayers to a larger extent than does TRIA. Solid-state ³¹P magic-angle spinning nuclear magnetic resonance spectra of LUVs showed two chemical shifts, at 0.009 and −11.988 ppm, at low temperatures (20°C), while at increasing temperatures (20–60°C) only one (at −11.988 ppm) was prominent and the other (0.009 ppm) gradually became obscure. However, LUVs with TRIA exhibited only one of the shifts at 0.353 ppm even at lower temperatures and JA did not affect the chemical shifts. An erratum to this article can be found at     

Effect of fluorescence quenching model on quinoline derivative with cobalt chloride metal ion using steady state and time resolved spectroscopic methods

Quinoline derivative, i.e. quinilone yellow with the scientific name [sodium 2-(2,3-dihydro-1,3-dioxo-1H-inden-2-yl)quinoline-6,8-disulphonate] (SQDS) is analysed for fluorescence resonance energy transfer (FRET). Fluorescence quenching mechanism is studied by employing steady state and transient state spectroscopic measurements. Cobalt chloride is used as quencher in the present study. Linearity was observed in Stern–Volmer plots for transient state as well as steady state. This was further attributed to a mechanism of collisional quenching. Efficiency in fluorescence quenching is observed as there is a correlation between quenching constants of both transient and steady state. A significant energy transfer is reported between metal ions and SQDS molecule, according to FRET theory. Characterization results are studied and analysed. Application in the field of non-linear optics are predicted for SQDS. With Kurtz and Perry powder technique, SHG (second harmonic generation) efficiency was measured using Q-switched mode locked Nd:YAG laser emitting 1064 nm the first time with this compound.     

Comparison of binding interactions of lomefloxacin to serum albumin and serum transferrin by resonance light scattering and fluorescence quenching methods

The interaction between lomefloxacin (LMF) and two drug carrier proteins, human serum albumin (HSA) and serum transferrin (TF), were studied and compared by fluorescence quenching, resonance light scattering (RLS), and circular dichroism (CD) spectroscopic along with molecular modeling. Fluorescence data show that LMF has a stronger quenching effect on HSA than on TF. The binding constant and the number of binding sites were calculated as 6.00 x 10(5) M(-1) and 0.77 for HSA, and 4.66 x 10(5) M(-1) and 1.02, for TF, respectively. Also, these binding parameters were calculated by RLS data, as a novel approach and were compared to that obtained from fluorescence. The micro-environment changes of Trp residues were evident in both proteins. The quantitative analysis of the secondary structure in both proteins further confirmed the drug-induced conformational changes. The distance (r) between donors (HSA and TF) and acceptor (LMF) were obtained by fluorescence resonance energy transfer (FRET) theory and found to be 1.83 nm and 1.71 nm for HSA and TF respectively. Moreover, molecular modeling studies suggested the sub-domain IB in HSA and N-lobe in TF as the candidate place for the formation of the binding site of LMF on these proteins.     

Human serum albumin–malathion complex study in the presence of silver nanoparticles at different sizes by multi spectroscopic techniques

The binding of malathion to human serum albumin (HSA) in the presence of silver nanoparticles (AgNPs) was investigated for the first time by multiple spectroscopic methods such as fluorescence quenching, fluorescence resonance energy transfer (FRET), circular dichroism, red-edge excitation shift (REES), synchronous fluorescence and three dimensional fluorescence spectroscopy under physiological conditions .The results indicated that binding of malathion to HSA induced fluorescence quenching through static mechanism. The number of binding sites was calculated by double logarithmic equation. Changes in the micro-environment of the fluorophore residues were also probed by synchronous fluorescence spectroscopy and REES. Changes of secondary structure of HSA in HSA–malathion complex was verified by circular dichroism approach in the presence of AgNPs that showed the electrostatic interaction changes in the protein structure. The binding average distance (r) between the donor (HSA) and the acceptor (malathion) was measured and found to be 1.63?nm according to the Forster’s theory of non-radiation energy transfer which was <7?nm confirmed the existence of static quenching in the presence of AgNPs. The conformational changes of HSA by three-dimensional fluorescence spectroscopy were studied. By comparing the resonance light scattering in the binary and ternary systems, we could estimate the effect of AgNPs on the precipitation of the malathion on the HSA. Generally we have discussed the toxicity reduction effect of malathion in food industrial by the results of spectroscopy techniques.     

Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.     

Origin of laurdan sensitivity to the vesicle-to-micelle transition of phospholipid-octylglucoside system: a time-resolved fluorescence study

The fluorescent probe laurdan has been shown to be sensitive to the vesicle-to-micelle transition of phosphatidylcholine/octylglucoside (M. Paternostre, O. Meyer, C. Grabielle-Madelmont, S. Lesieur, and, Biophys. J. 69:2476-2488). On the other hand, a study on the photophysics of laurdan in organic solvents has shown that the complex de-excitation pathway of the probe can be described by two successive processes, i.e., an intramolecular charge transfer followed by dielectric relaxation of the solvent if polar. These two excited-state reactions lead to three emitting states, i.e., a locally excited state, a charge transfer state, and a solvent relaxed state (M. Viard, J. Gallay, M. Vincent, B. Robert and, Biophys. J. 73:2221-2234). Experiments have been performed using time-resolved fluorescence on the probe inserted in amphiphile aggregates (mixed liposomes, mixed micelles) different in detergent-to-lipid ratios. The results have been compared with those obtained for laurdan inserted in dipalmitoyl phosphatidylcholine liposomes in the gel and in the fluid lamellar phase. Except for laurdan in dipalmitoyl phosphatidylcholine liposomes in the gel lamellar phase, the red part of the emission spectra originates from the de-excitation of the relaxed excited state of laurdan, indicating that indeed the dielectric relaxation process is an important phenomena in the ground-state return pathway of this probe. On the other hand, the maximization entropy method (MEM) analysis of the fluorescence decay recorded in the blue part of the emission spectra indicates that the dielectric relaxation is not the only reaction occurring to the excited state of laurdan. Moreover, the analysis of the fluorescence decays of laurdan inserted in gel lamellar dipalmitoylphosphatidylcholine (DPPC) liposomes indicates excited-state reactions, although dielectric relaxation is impossible. These results are in agreement with the de-excitation pathway determined from laurdan behavior in organic solvent even if, in most of the aggregates studied in this work, the major phenomenon is the dielectric relaxation of the solvent. All along the vesicle-to-micelle transition, we have observed that the lifetime of the relaxed excited state of laurdan continuously decreases probably due to a dynamic quenching process by water molecules. On the other hand, the time constant of the dielectric relaxation process remains almost unchanged in the lamellar part of the transition but abruptly decreases as soon as the first mixed micelle is formed. This decrease is continuous all over the rest of the transition even if it is more pronounced in the mixed liposomes' and mixed micelles' coexistence. The increase of the octylglucoside-to-lipid ratio of the mixed micelles via the change of the size and the shape of the aggregates may facilitate the penetration and the mobility of water molecules. Therefore, during the vesicle-to-micelle transition, laurdan probes the evolution of both the amphiphile packing in the aggregates and the increase of the interface polarity. This study finally shows that the detergent-to-lipid ratio of the mixed micelles is an important parameter to control to limit the penetration and the mobility of water within the amphiphile aggregates and that laurdan is a nice tool to monitor this phenomenon.     

Binding of teicoplanin and vancomycin to bovine serum albumin in vitro: a multispectroscopic approach and molecular modeling

In this paper, the binding properties of teicoplanin and vancomycin to bovine serum albumin (BSA) were investigated using fluorescence quenching, synchronous fluorescence, Fourier transform infrared (FTIR), circular dichroism (CD) and UV–vis spectroscopic techniques and molecular docking under simulative physiological conditions. The results obtained from fluorescence quenching data revealed that the drug–BSA interaction altered the conformational structure of BSA. Meanwhile, the 3D fluorescence, CD, FTIR and UV–vis data demonstrated that the conformation of BSA was slightly altered in the presence of teicoplanin and vancomycin, with different reduced α‐helical contents. The binding distances for the drug–BSA system were provided by the efficiency of fluorescence resonance energy transfer (FRET). Furthermore, the thermodynamic analysis implied that hydrogen bond and van der Waals' forces were the main interaction for the drug–BSA systems, which agreed well with the results from the molecular modeling study. The results obtained herein will be of biological significance in future toxicological and pharmacological investigation. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of ionic strength on the organization and dynamics of tryptophan residues in erythroid spectrin: a fluorescence approach

The ionic strength of the medium plays an important role in the structure and conformation of erythroid spectrin. The spectrin dimer is a flexible rod at physiological ionic strength. However, lower ionic strength results in elongation and rigidification (stiffening) of spectrin as shown earlier by electron microscopy and hydrodynamic studies. The ionic strength induced structural transition does not involve any specific secondary structural changes. In this article, we have used a combination of fluorescence spectroscopic approaches that include red edge excitation shift (REES), fluorescence quenching, time-resolved fluorescence measurements, and chemical modification of the spectrin tryptophans to assess the environment and dynamics of tryptophan residues of spectrin under different ionic strength conditions. Our results show that while REES, fluorescence anisotropy, lifetime, and chemical modification of spectrin tryptophans remain unaltered in low and high ionic strength conditions, quenching of tryptophan fluorescence by the aqueous quencher acrylamide (but not the hydrophobic quencher trichloroethanol) and resonance energy transfer to a dansyl-labeled fatty acid show differences in tryptophan environment. These results, which report tertiary structural changes in spectrin upon change in ionic strength, are relevant in understanding the molecular details underlying the conformational flexibility of spectrin.     

Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer.

We measured the nonradiative fluorescence resonance energy transfer between 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled lipids (amine labeled phosphatidylethanolamine or acyl chain labeled phosphatidylcholine) and rhodamine labeled lipids in large unilamellar dioleoylphosphatidylcholine vesicles. Two new rhodamine labeled lipid analogues, one a derivative of monolauroylphosphatidylethanolamine and the other of sphingosylphosphorylcholine, were found to exchange through the aqueous phase between vesicle populations but not to be capable of rapid transbilayer movement between leaflets. Energy transfer from NBD to rhodamine was measured using liposomes with symmetric or asymmetric distributions of these new rhodamine labeled lipid analogues to determine the relative contributions of energy transfer between donor and acceptor fluorophores in the same (cis) and opposite (trans) leaflets. Since the characteristic R0 values for energy transfer ranged from 47 to 73 A in all cases, significant contributions from both cis and trans energy transfer were observed. Therefore, neither of these probes acts strictly as a half-bilayer quencher of NBD lipid fluorescence. The dependence of transfer efficiency on acceptor density was fitted to a theoretical treatment of energy transfer to determine the distances of closest approach for cis and trans transfer. These parameters set limits on the positions of the fluorescent groups relative to the bilayer center, 20-31 A for NBD and 31-55 A for rhodamine, and provide a basis for future use of these analogues in measurements of transbilayer distribution and transport.     

Development of a fluorescent substrate to measure hyaluronidase activity

A novel fluorescent substrate (termed FRET-HA) to quantitatively assess hyaluronidase activity was developed. Hyaluronan (HA), the major substrate for hyaluronidase, was dual labeled with fluorescein amine and rhodamine B amine. The fluorescein amine fluorescence signal was significantly quenched and the rhodamine B amine signal was significantly enhanced due to fluorescence resonance energy transfer (FRET). In the presence of bovine testes hyaluronidase, cleavage of HA disrupted FRET, resulting in a loss of the fluorescein amine quenching that was dependent on both enzyme concentration and time. Increase in the fluorescein amine signal could be conveniently monitored in both noncontinuous and continuous fashions. The K_m value for bovine testes hyaluronidase was determined using FRET-HA in a continuous fluorescent assay. Importantly, the estimated K_m value for bovine testes hyaluronidase using FRET-HA as the substrate was in excellent agreement with K_m values reported previously for this enzyme using native (i.e., unlabeled) HA. Therefore, FRET-HA is a reliable substrate for quantitatively assessing the HA/hyaluronidase molecular interaction. The simplicity, sensitivity, and versatility of the FRET-HA substrate suggest that it will have utility in a variety of assay platforms and should be a new tool for assessing hyaluronidase activity.     

FRET detection of cellular alpha4-integrin conformational activation

Integrins are cell adhesion receptors, expressed on every cell type, that have been postulated to undergo conformational changes upon activation. Here, different affinity states were generated by exposing alpha4-integrins to divalent ions or by inside-out activation using a chemokine receptor. We probed the dynamic structural transformation of the integrin on live cells using fluorescence resonance energy transfer (FRET) between a peptide donor, which specifically binds to the alpha4-integrin, and octadecyl rhodamine B acceptors incorporated into the plasma membrane. We analyzed the data using a model that describes FRET between a random distribution of donors and acceptors in an infinite plane. The distance of closest approach was found to vary with the affinity of the integrin. The change in distance of closest approach was approximately 50 A between resting and Mn2+ activated receptors and approximately 25 A after chemokine activation. We used confocal microscopy to probe the lateral organization of donors and acceptors subsequent to integrin activation. Taken together, FRET and confocal results suggest that changes in FRET efficiencies are primarily due to the vertical extension of the integrin. The coordination between the extension of alpha4-integrin and its affinity provides a mechanism for Dembo's catch-bond concept.     

Structure-based calculation of multi-donor multi-acceptor fluorescence resonance energy transfer in the 4×6-mer tarantula hemocyanin

Hemocyanins are oxygen carriers of arthropods and molluscs. The oxygen is bound between two copper ions, forming a Cu(II)-O₂ ^2–-Cu(II) complex. The oxygenated active sites create two spectroscopic signals indicating the oxygen load of the hemocyanins: first, an absorption band at 340 nm which is due to a ligand-to-metal charge transfer complex, and second, a strong quenching of the intrinsic tryptophan fluorescence, the cause of which has not been definitively identified. We showed for the 4×6-mer hemocyanin of the tarantula Eurypelma californicum that the fluorescence quenching of oxygenated hemocyanin is caused exclusively by fluorescence resonance energy transfer (FRET). The tarantula hemocyanin consists of 24 subunits containing 148 tryptophans acting as donors and 24 active sites as acceptors. The donor–acceptor distances are determined on the basis of a closely related crystal structure of the horseshoe crab Limulus polyphemus hemocyanin subunit II (68–79% homology). Calculation of the expected fluorescence quenching and the measured transfer efficiency coincided extraordinary well, so that the fluorescence quenching of oxygenated tarantula hemocyanin can be completely explained by Förster transfer. This results explain for the first time, on a molecular basis, why fluorescence quantum yield can be used as an intrinsic signal for oxygen load of at least one arthropod hemocyanin, in particular that from the tarantula.     

Photophysics of metalloazurins

The fluorescence lifetimes of Cu(II), Cu(I), Ag(I), Hg(II), Co(II), and Ni(II) azurin Pae from Pseudomonas aeruginosa and Cu(II), Cu(I), and Hg(II) azurin Afe from Alcaligenes faecalis were measured at 295 K by time-correlated single-photon counting. In addition, fluorescence lifetimes of Cu(II) azurin Pae were measured between 30 and 160 K and showed little change in value. Ultraviolet absorption difference spectra between metalloazurin Pae and apoazurin Pae were measured, as were the fluorescence spectra of metalloazurins. These spectra were used to determine the spectral overlap integral required for dipole-dipole resonance calculations. All metalloazurins exhibit a reduced fluorescence lifetime compared to their respective apoazurins. Forster electronic energy transfer rates were calculated for both metalloazurin Pae and metalloazurin Afe derivatives; both enzymes contain a single tryptophyl residue which is located in a different position in the two azurins. These azurins have markedly different fluorescence spectra, and electronic energy transfers occur from these two tryptophyl sites with different distances and orientations and spectral overlap integral values. Intramolecular distances and orientations were derived from an X-ray crystallographic structure and a molecular dynamic simulation of the homologous azurin Ade from Alcaligenes denitrificans, which contains both tryptophyl sites. Assignments were made of metal-ligand-field electronic transitions and of transition dipole moments and directions for tryptophyl residues, which accounted for the observed fluorescence quenching of Hg(II), Co(II), and Ni(II) azurin Pae and Cu(II) and Hg(II) azurin Afe. The fluorescence of azurin Pae is assigned as a 1Lb electronic transition, while that of azurin Afe is 1La. The marked fluorescence quenching of Cu(II) azurin Pae and Cu(I) azurin Pae and Afe is less well reproduced by our calculations, and long-range oxidative and reductive electron transfer, respectively, are proposed as additional quenching mechanisms. This study illustrates the application of Forster electronic energy transfer calculations to intramolecular transfers in structurally well characterized molecular systems and demonstrates its ability to predict observed fluorescence quenching rates when the necessary extensive structural, electronic transition assignment, and spectroscopic data are available. The agreement between Forster calculations and quenching rates derived from fluorescence lifetime measurements suggests there are limited changes in conformation between crystal structure and solution structures, with the exception of the tryptophyl residue of azurin Afe, where a conformation derived from a molecular simulation in water was necessary rather than that found in the crystal structure.     

A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein.

A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein (LDL) particles in living cells has been developed. The method is based on the fluorescence resonance energy transfer (RET) between two fluorescent molecules incorporated into LDL particles. NBD-cholesterol linoleate (NBD-CL) and octadecyl rhodamine B (R18) were incorporated simultaneously into LDL, as a RET donor and a RET acceptor, respectively. In this preparation of LDL (RET-LDL), efficient RET was observed, and after the disruption of the LDL particle by a Triton X-100 treatment, the relief of the RET was observed. RET-LDL was endocytosed by CHO cells via LDL receptors, and the RET-LDL particles were disintegrated after the uptake. The resultant relief of the RET upon the disintegration of the LDL was monitored by flow cytometry, and the amount of intact LDL in cells was estimated by calculation. The disintegration occurred with an about 25 min lag, and was inhibited by several lysosomal inhibitors. These results indicate that the disintegration was not a nonspecific event, but took place at the level of lysosomes. Since living cells can be analyzed by the present method, when coupled to flow sorting, it would permit the isolation of cells having different properties in the endocytic pathway of LDL.     

A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes

A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.