共查询到20条相似文献,搜索用时 15 毫秒
1.
N L Vekshin 《Journal of biochemical and biophysical methods》1987,15(2):97-104
The movement of pyrene in a lipid bilayer is shown to occur not only in the lateral but also transmembrane direction. Within the excited state lifetime, the pyrene monomer elevates from the depth to the polar region of the membrane and emits a luminescence photon. The excimer does not exhibit any marked transmembrane movement. The luminescence quenching efficiency of monomers and excimers depends on the depth of penetration of the quencher into the membrane. In the lipid bilayer, pyrene luminescence is strongly quenched by oxygen. The binding of pyrene to membrane proteins protects it from quenching. It has been concluded that the widely used estimations of membrane viscosity from pyrene luminescence intensity are incorrect. 相似文献
2.
The pyrene movement in a lipid bilayer has been shown to occur not only in the lateral but also transmembrane direction. Within the excited state lifetime the pyrene monomer elevates from the depth to the polar regions of the membrane and emits a luminescence photon. The excimer does not exhibit any marked transmembrane movement while luminescing from the hydrophobic regions. The luminescence quenching efficiency of monomers and excimers depends on the depth of quencher penetration into the membrane. In the lipid bilayer the pyrene luminescence is strongly quenched by molecular oxygen. The pyrene binding to membrane proteins protects it from quenching. A conclusion has been made that the carrying out estimations of membrane viscosity from pyrene luminescence require considerable correction. 相似文献
3.
The transfer of pyrene between 1-acid glycoprotein, acethylcholinesterase and sonicated liposomes was used to monitor glycoprotein-protein interaction on the lipid bilayer. When a density solution of glycoprotein or protein labeled with pyrene was mixed with unlabeled suspension of free-phospholipid liposomes, or suspensions containing the complexes of glycoprotein-lipid, protein-lipid, or glycoprotein-protein-lipid, pyrene excimer fluorescence increased with a half-time of approximately 30–50 msec. Since the increase in excimer fluorescence indicates an increase in the microscope concentrations of pyrene, the observed fluorescence change reflects pyrene transfer. The half-times for the increase in excimer fluorescence were determined in the presence of glycoprotein and protein in the liposomes. On the basis of the determined half-times it was concluded that both, glycoprotein and protein are bound on the lipid bilayer. Our data also suggest that the thickness of the lipid bilayer is significantly changed in this case. The observation suggests strongly that the limiting step in the transfer of pyrene is not the dissociation of pyrene, but the uptake of the pyrene monomers by the lipid phase. 相似文献
4.
The aldehydes present in acid-soluble type I collagen react with pyrenebutyrylhydrazine to form various types of complexes under different reaction conditions. These complexes exhibit one or more of three different pyrene fluorescence bands: monomer, excimer, and aggregate fluorescence. Collagen, whose aldehydes have been reduced with NaBH4, does not react with this fluorescent hydrazine, confirming that the hydrazine reacts specifically with aldehyde groups to form hydrazones. The absence of a reaction with pepsin-treated collagen also shows that the fluorescent labels are primarily in the nonhelical terminal telopeptides. Upon dialysis, the pyrene label bound to a saturated aldehyde in an α-chain is lost; whereas that bound to an unsaturated aldehyde remains on the protein. The pyrene monomer fluorescence in the β-chain of old collagen is stronger than that of young collagen. The formation of the pyrene excimer fluorescence implies the proximity of two pyrene molecules, probably attached to two adjacent aldehydes. Upon changing from acidic to neutral pH, both excimer and aggregate fluorescence bands disappear within a few seconds, revealing a very rapid alteration at the telopeptides. 相似文献
5.
The presence of proteins in lipid bilayers always decreases the excimer formation rate of pyrene and pyrene lipid analogues
in a way that is related to the protein-to-lipid ratio. Energy transfer measurements from intrinsic tryptophans to pyrene
have shown (Engelke et al., 1994), that in microsomal membranes, the excimer formation rate of pyrene and pyrene fatty acids
is heterogeneous within the membrane plane, because a lipid layer of reduced fluidity surrounds the microsomal proteins. This
study investigates whether of not liposomes prepared from egg yolk phosphatidylcholine with incorporated gramicidin A give
results comparable to those from microsomal membranes. The results indicate that the influence of proteins on the lipid bilayer
cannot be described by one unique mechanism: Small proteins such as gramicidin A obviously reduce the excimer formation rate
by occupying neighboring positions of the fluorescent probe and thus decrease the pyrene collision frequency homogeneously
in the whole membrane plane, while larger proteins are surrounded by a lipid boundary layer of lower fluidity than the bulk
lipid.
The analysis of the time-resolved tryptophan fluorescence of gramicidin A incorporated liposomes reveals, that the tryptophan
quenching by pyrene is stronger for tryptophans located closely below the phospholipid headgroup region because of the pyrene
enrichment in this area of the lipid bilayer.
Received: 29 December 1996/Revised: 15 May 1996 相似文献
6.
Manduca sexta apolipophorin III (apoLp-III), an 18-kDa, monomeric, insect hemolymph apolipoprotein, is comprised of five amphipathic alpha-helices arranged as a globular bundle in the lipid-free state. Upon lipid binding, it is postulated that the bundle opens, exposing a continuous hydrophobic surface which becomes available for lipid interaction. To investigate lipid binding-induced helical rearrangements, we exploited the unique fluorescence characteristics of N-(1-pyrene)maleimide. Pyrene is a spatially sensitive extrinsic fluorescent probe, which forms excited-state dimers (excimers) upon close encounter with another pyrene molecule. Cysteine residues were introduced into apoLp-III (which otherwise lacks cysteine) at Asn 40 (helix 2) and/or Leu 90 (helix 3), creating two single-cysteine mutants (N40C-apoLp-III and L90C-apoLp-III) and N40C/L90C-apoLp-III, a double-cysteine mutant, which were labeled with pyrene maleimide. Pyrene-labeled N40C/L90C-apoLp-III, but not the pyrene-labeled single-cysteine mutants, exhibited strong excimer fluorescence in the lipid-free, monomeric state. Guanidine hydrochloride titration and temperature studies revealed a loss in excimer fluorescence, accompanied by a loss in the molar ellipticity of the protein. When apoLp-III interacts with phospholipid vesicles to form disklike complexes, a significant loss in excimer fluorescence was noted, indicating that the helices bearing the pyrene moieties diverge from each other. Pyrene excimer fluorescence was further employed to examine the relative orientation of lipid-bound apoLp-III molecules. Pyrene-labeled N40C- or L90C-apoLp-III displayed no excimer fluorescence in the disk complexes, while complexes prepared with an equal mixture of both single-labeled mutants did emit excimer fluorescence, indicating apoLp-III adopts a preferred nonrandom orientation around the perimeter of the bilayer disk. These studies establish pyrene excimer fluorescence as a useful spectroscopic tool to address intra- and intermolecular interactions of exchangeable apolipoproteins upon binding to lipid. 相似文献
7.
Kostenko E Dobrikov M Pyshnyi D Petyuk V Komarova N Vlassov V Zenkova M 《Nucleic acids research》2001,29(17):3611-3620
Oligonucleotide conjugates bearing two pyrene residues attached to 5′-phosphate through a phosphoramide bond were synthesised. Fluorescence spectra of the conjugates show a peak typical of monomer emission (λmax 382 nm) and a broad emission peak with λmax 476 nm, which indicates the excimer formation between the two pyrene residues. Conjugation of these two pyrene residues to the 5′-phosphate of oligonucleotides does not affect the stabilities of heteroduplexes formed by conjugates with the corresponding linear strands. A monomer fluorescence of the conjugates is considerably affected by the heteroduplex formation allowing the conjugates to be used as fluorescent hybridisation probes. The 5′-bis-pyrenylated oligonucleotides have been successfully used for investigation of affinity and kinetics of antisense oligonucleotides binding to the multidrug resistance gene 1 (PGY1/MDR1) mRNA. The changes of excimer fluorescence of the conjugates occurring during hybridisation depended on the structure of the binding sites: hybridisation to heavily structured parts of RNA resulted in quenching of the excimer fluorescence, while binding to RNA regions with a loose secondary structure was accompanied by an enhancement of the excimer fluorescence. Potentially, these conjugates may be considered as fluorescent probes for RNA structure investigation. 相似文献
8.
Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy
Martí AA Li X Jockusch S Li Z Raveendra B Kalachikov S Russo JJ Morozova I Puthanveettil SV Ju J Turro NJ 《Nucleic acids research》2006,34(10):3161-3168
We report here the design, synthesis and application of pyrene binary oligonucleotide probes for selective detection of cellular mRNA. The detection strategy is based on the formation of a fluorescent excimer when two pyrene groups are brought into close proximity upon hybridization of the probes with the target mRNA. The pyrene excimer has a long fluorescence lifetime (>40 ns) compared with that of cellular extracts (~7 ns), allowing selective detection of the excimer using time-resolved emission spectra (TRES). Optimized probes were used to target a specific region of sensorin mRNA yielding a strong excimer emission peak at 485 nm in the presence of the target and no excimer emission in the absence of the target in buffer solution. While direct fluorescence measurement of neuronal extracts showed a strong fluorescent background, obscuring the detection of the excimer signal, time-resolved emission measurements indicated that the emission decay of the cellular extracts is ~8 times faster than that of the pyrene excimer probes. Thus, using TRES of the pyrene probes, we are able to selectively detect mRNA in the presence of cellular extracts, demonstrating the potential for application of pyrene excimer probes for imaging mRNAs in cellular environments that have background fluorescence. 相似文献
9.
The effect of ionic strength on the fluidity of rabbit intestinal brush-border membranes has been studied using two fluorescence probes, pyrene and 1-anilino-8-naphthalene sulfonate (ANS). The imposition of a potential gradient on the pyrene-probed membrane vesicles () with increasing NaCl concentration in the medium resulted in a marked enhancement of the excimer formation efficiency, accompanied by a decrease in the ratio of fluorescence intensities of the probe at 392 and 375 nm. Fluorescence polarization of the pyrene-membrane complex is independent of temperature in the absence of salts, while it is dependent on temperature from 10 to 47°C in the presence of salts, as shown by the thermal Perrin plots of polarization. It has been demonstrated that there is a linear relationship between the changes in the pyrene excimer formation efficiency in the membranes and of the values of the binding parameters of ANS for the membranes. From these results, it is suggested that the lipid phase of the membranes becomes more fluid by shielding negatively charged groups of the membrane surface and that there is a fairly close correlation between the membrane organization and the membrane surface charge density. 相似文献
10.
Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.
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In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase. 相似文献
11.
A method for estimating the fluidity of natural membranes from the pyrene excimer/monomer fluorescence ratio (Ie/Im) is proposed. The method makes it possible to exclude artefacts such as fluorescence quenching, aggregation, and redistribution of the probe in lipid mains with different microviscosity. It is shown that, upon variation of intramembrane pyrene concentration [pyr], the occurrence of a common crossover point in pyrene fluorescence spectra normalized to the corresponding probe concentration (isoemission or isobestic point) or, as a consequence, the linear dependence of Ie/[pyr] on Im/[pyr] can serve as a criterion of diffusion (fluidity)-controlled excimerization of pyrene. The isobestic point can be used for determining the range of working concentrations of the probe in membrane suspension. It was found from the intensity of pyrene fluorescence in the isobestic point and quenching with potassium iodide that at t < 30 degrees C, the probe is uniformly distributed throughout the membrane, and its excimerization is mainly controlled by the microviscosity of environment. 相似文献
12.
The effect of ionic strength on the fluidity of rabbit intestinal brush-border membranes has been studied using two fluorescence probes, pyrene and 1-anilino-8-naphthalene sulfonate (ANS). The imposition of a potential gradient on the pyrene-probed membrane vesicles (out greater than in) with increasing NaCl concentration in the medium resulted in a marked enhancement of the excimer formation efficiency, accompanied by a decrease in the ratio of fluorescence intensities of the probe at 392 and 375 nm. Fluorescence polarization of the pyrene-membrane complex is independent of temperature in the absence of salts, while it is dependent on temperature from 10 to 47 degrees C in the presence of salts, as shown by the thermal Perrin plots of polarization. It has been demonstrated that there is a linear relationship between the changes in the pyrene excimer formation efficiency in the membranes and of the values of the binding parameters of ANS for the membranes. From these results, it is suggested that the lipid phase of the membranes becomes more fluid by shielding negatively charged groups of the membrane surface and that there is a fairly close correlation between the membrane organization and the membrane surface charge density. 相似文献
13.
M. Ollmann A. Robitzki G. Schwarzmann H.-J. Galla 《European biophysics journal : EBJ》1988,16(2):109-112
We have investigated the effect of bulk viscosity on lipid translational diffusion using the excimer formation technique. In contrast to a study by Vaz et al. (1987), performed with the fluorescence recovery after photobleaching technique, we observed only a minor decrease of less than a factor of two for pyrene labelled phosphatidylcholine in glycerinated phosphatidylcholine bilayer membranes compared to an aqueous dispersion. Even the diffusion of pyrene labelled gangliosides with an oligosaccharide head-group that protrudes from the membrane surface is not strongly restricted by the increased bulk viscosity. We conclude that the viscosity of the fluid bounding the lipid bilayers is of minor importance for the diffusion of membrane lipids.Abbreviations DPPC
1-2 dipalmitoyl-sn-glycero-3-phosphocholine
- DSPC
1-2 distearoyl-sn-glycero-3-phosphocholine
- PyPC
1-acyl-2-[10(-1-pyrene)decanoyl]-sn-glycero-3-phosphocholine
- PyGM1
N-12-(1-pyrene)dodecanoyl-lyso GM1
- PyGM2
N-12-(1-pyrene)dodecanoyl-lyso GM2
- PyGM3
N-12-(1-pyrene) dodecanoyl-lyso GM3
- IM
fluorescence intensity of the monomeric pyrene probe
- ID
fluorescence intensity of the excimer 相似文献
14.
In order to determine the microviscosity of human erythrocyte membrane suspensions, a method has been developed which is based on pyrene excimer formation. First, measurements of partitioning of pyrene into membranes, in conjunction with known values for the volume of the lipid compartment of erythrocyte ghosts are used to determine the concentration of pyrene in the membrane lipid. Secondly, reported measurements of the diffusion constants of aromatic hydrocarbons similar in structure to pyrene, are used to derive an empirical equation relating solvent viscosity and the diffusion constant of pyrene. Then, measurements of pyrene excimer formation in a series of solvents ranging up to several poise in viscosity are used to determine that the interaction diameter of the excimer formation reaction is 3 +/- 1 A. Finally all these data are brought together in order to conclude that the viscosity of the lipid in the human erythrocyte ghost is 8.0, 4.0 and 1.6 P at 10, 25 and 40 degrees C, respectively. 相似文献
15.
Kinetic and steady-state measurements of pyrene fluorescence in a variety of model membranes are evaluated in terms of the theory of collisional excimer formation. In the region of 10(-3)-0.1 M pyrene, molecular fluorescence decay in membranes is biphasic and the two component lifetimes do not depend on the pyrene concentration. The lifetime data are consistent with the rate constant for collisional excimer formation being of the order 10(6) M-1 X s-1 or less. The concentration dependence of the component amplitudes is inconsistent with the theory of collisional excimer formation and suggests that pyrene exists in two forms in membranes: a slowly diffusing monomeric form and an aggregated form. The component of molecular fluorescence decay associated with aggregated pyrene is highly correlated with steady-state excimer fluorescence, suggesting that excimer fluorescence in membranes arises from aggregated pyrene in which excimers are formed by a static rather than a collisional mechanism. It is suggested that the concentration dependence of excimer to molecular fluorescence intensity ratios in membranes is related to the equilibrium constant for exchange between monomeric and aggregated pyrene forms rather than to the collisional excimer formation rate constant. 相似文献
16.
Using a helix-loop-helix construct consisting of the adjacent transmembrane segments 3 and 4 of the cystic fibrosis transmembrane conductance regulator (CFTR) labeled with pyrene at both N and C termini, we describe a system for the study of intramolecular helix-helix interactions within a polytopic membrane protein. Through measurement of pyrene excimer band intensity as a determinant of helix-helix proximity, we show that the helices retain tertiary contacts in detergent micelles. Notably, the nature of the micellar detergent can alter the stability of these contacts, with perfluorooctanoate highly supportive, lysophosphatidylcholine and lysophosphatidylglycerol somewhat less tolerant, and SDS largely intolerant of such interactions. This construct is further employed to study the role of the acyl chain length of micellar detergents in modulating interhelical packing; detergents having acyl chains of 9 carbons display the greatest extent of helical packing. These results provide important information regarding the role of lipids on membrane protein folding and conformation as well as demonstrate the usefulness of a pyrene-based system in studying the forces that govern interhelical packing. 相似文献
17.
Gary R. Burleson Charles F. Kulpa Haydn E. Edwards J. Kerry Thomas 《Experimental cell research》1978,116(2):291-300
The fluorescent probes pyrene, pyrene butyric acid and N-phenyl 1-naphthylamine were used to study membranes of normal cells, RSV-transformed cells, cells treated with a proteolytic enzyme, and cells persistently infected with lymphocytic choriomeningitis virus. The lifetimes of excited pyrene and pyrene butyric acid showed only minor changes when these probes were in normal, transformed, trypsinized or persistently infected cells. However, pyrene, but not pyrene butyric acid, lifetimes are shorter in cell membranes than in homogeneous solvents. The quenching of excited pyrene in cells by quencher molecules was slower than corresponding reactions in homogeneous solutions indicating that the probe was screened from the quenchers by the membrane. However, quenching reactions with the pyrene butyric acid probe were similar in cells and homogeneous solvents. This indicates that pyrene and pyrene butyric acid reside in different lipid regions of the membrane. Transformed and trypsinized cells showed increased membrane fluidity compared to normal and persistently infected cells. Membrane fluidity was determined from the excimer/monomer fluorescence ratios of pyrene, and by the polarization of N-phenyl 1-naphthylamine fluorescence. Several techniques distinguished between normal and transformed or trypsinized cells; however, the only parameter unique to viral transformation was a blue shift of the fluorescence maxima of N-phenyl 1-naphthylamine. This shift reflected a less polar environment for N-phenyl 1-naphthylamine in virus-transformed cells. 相似文献
18.
Tetherin/Bst-2 is a cell surface protein that can act as a restriction factor against a number of enveloped viruses, including HIV-1. It acts by tethering new virus particles to the host cell membrane, promoting their internalization and degradation. Tetherin is a type II membrane protein, with an N-terminal transmembrane domain, an extracellular coiled-coil domain, and a C-terminal GPI anchor. This double membrane anchor is important for anti-HIV activity, as is dimerization of the coiled-coil domain, but despite recent crystal structures of the coiled-coil ectodomains of human and mouse tetherin, the topology of tetherin with respect to host and viral membranes has yet to be determined. The tetherin transmembrane domain is also thought to mediate interactions with the HIV-1 encoded integral membrane protein Vpu, which is an antagonist of tetherin, through direct binding to the transmembrane region of Vpu. Using a combination of SDS-PAGE, size exclusion chromatography, and pyrene excimer fluorescence, we show that in the absence of the coiled-coil domain the transmembrane domain of human tetherin forms parallel homodimers in membrane mimetic environments. Transmembrane domain dimerization does not require disulfide bond formation and is favored in TFE, SDS micelles, and POPC liposomes. This observation has implications for functional models of tetherin, suggesting that both transmembrane domains in the dimeric molecule are inserted into the same lipid bilayer, rather than into opposing membranes. 相似文献
19.
Kok Hong Lim Heng Huang Arnd Pralle Sheldon Park 《Biotechnology and bioengineering》2013,110(1):57-67
The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (Kd = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (Tm = 59.8°C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction. Biotechnol. Bioeng. 2013; 110: 57–67. © 2012 Wiley Periodicals, Inc. 相似文献
20.
The intensities of fluorescence emission for pyrene and a number of its derivatives increase on binding to lipid bilayers and to the (Ca2+-Mg2+)-ATPase purified from rabbit muscle sarcoplasmic reticulum. The effect is particularly marked for the less water-soluble derivatives. Changes in intensity for monomer and excimer emission as a function of lipid concentration can be fitted to a simple model to obtain binding parameters. The number of binding sites per lipid is 0.2-0.4. For the ATPase system, at least two classes of sites are necessary to fit the data, one corresponding to the lipid component and one to sites on the ATPase. Excimer emission from the postulated sites on the ATPase is less marked than that from lipid. Pyrene-dodecanoic acid and pyreneundecyltrimethylammonium bromide, which bind to a large number of sites on the ATPase, cause marked inhibition of ATPase activity at high concentration. Pyrene and a number of water-soluble derivatives cause stimulation of the ATPase reconstituted with dimyristoleoylphosphatidylcholine and little inhibition and bind to a small number of sites on the ATPase. It is concluded that excimer emission from pyrene derivatives in systems containing proteins cannot be used to obtain reliable information about rates of diffusion in the lipid component of the membrane. 相似文献