Detection of local polarity and conformational changes at the active site of rabbit muscle creatine kinase with a new arginine-specific fluorescent probe

A new polarity-sensitive fluorescent probe, 3-(4-chloro-6-p-glyoxal-phenoxy-1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine (CGTDP), is synthesized for selective labeling of active-site arginine residues. The probe comprises a neutral red moiety as a polarity-sensitive fluorophore and a phenylglyoxal unit as an arginine-specific labeling group. The probe exhibits a sensitive response of shift of fluorescence maximum emission wavelength to solvent polarity only instead of pH or temperature, which leads to the use of the probe in detecting the local polarity and conformational changes of the active site of rabbit muscle creatine kinase (CK) denatured by pH or temperature. The polarity of the active site domain has been first found to correspond to a dielectric constant of about 44, and the conformational change of the active site directly revealed by CGTDP occurs far before that of CK as a whole disclosed by the intrinsic tryptophan fluorescence during acid or thermal denaturation. The present strategy may provide a useful method to detect the local polarity and conformational changes of the active sites of many enzymes that employ arginine residues as anion recognition sites under different denaturation conditions.     

Potential charge transfer probe induced conformational changes of model plasma protein human serum albumin: Spectroscopic, molecular docking, and molecular dynamics simulation study

The nature of binding of specially designed charge transfer (CT) fluorophore at the hydrophobic protein interior of human serum albumin (HSA) has been explored by massive blue-shift (82 nm) of the polarity sensitive probe emission accompanying increase in emission intensity, fluorescence anisotropy, red edge excitation shift, and average fluorescence lifetimes. Thermal unfolding of the intramolecular CT probe bound HSA produces almost opposite spectral changes. The spectral responses of the molecule reveal that it can be used as an extrinsic fluorescent reporter for similar biological systems. Circular dichrosim spectra, molecular docking, and molecular dynamics simulation studies scrutinize this binding process and stability of the protein probe complex more closely.     

Characterization of local polarity and hydrophobic binding sites of beta-lactoglobulin by using N-terminal specific fluorescence labeling

Because of wide ligand-binding ability and significant industrial interest of beta-lactoglobulin (beta-LG), its binding properties have been extensively studied. However, there still exists a controversy as to where a ligand binds, since at least two potential hydrophobic binding sites in beta-LG have been postulated for ligand binding: an internal one (calyx) and an external one (near the N-terminus). In this work, the local polarity and hydrophobic binding sites of beta-LG have been characterized by using N-terminal specific fluorescence labeling combined with a polarity-sensitive fluorescent probe 3-(4-chloro-6-hydrazino- 1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine (CHTDP). The polarity within the calyx is found to be extremely low, which is explained in terms of superhydrophobicity possibly resulting from its nanostructure, and the polarity is increased with the destruction of the calyx by heat treatment. However, the polarity of the N-terminal domain in native beta-LG is decreased after thermal denaturation. This polarity trend toward decreasing instead of increasing shows that beta-LG may have no definite external hydrophobic binding site. The hydrophobic binding of a ligand such as CHTDP at the surface of the protein is probably achieved via appropriate assembling of corresponding hydrophobic residues rather than via a fixed external hydrophobic binding site. Also, the ligand-binding location in beta-LG is found to be relevant to not only experimental conditions (pH < or = 6.2 or pH > 7.1) but also binding mechanisms (hydrophobic affinity or electrostatic interaction).     

Interaction of Escherichia coli hemolysin with biological membranes. A study using cysteine scanning mutagenesis.

Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belonging to the family of RTX-toxins. Lytic activity depends on binding of Ca2(+) to the C-terminus of the molecule. The N-terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane-inserting domain. In this study, 13 HlyA cysteine-replacement mutants were constructed and labeled with the polarity-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan). The fluorescence emission of the label was examined in soluble and membrane-bound toxin. Binding effected a major blue shift in the emission of six residues within the N-terminal hydrophobic domain, indicating insertion of this domain into the lipid bilayer. The emission shifts occurred both in the presence and absence of Ca2(+), suggesting that Ca2(+) is not required for the toxin to enter membranes. However, binding of Ca2(+) to HlyA in solution effected conformational changes in both the C-terminal and N-terminal domain that paralleled activation. Our data indicate that binding of Ca2(+) to the toxin in solution effects a conformational change that is relayed to the N-terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.     

Calcium-triggered membrane interaction of the alpha-synuclein acidic tail

Alpha-synuclein (alpha-syn) is a 140-residue protein that aggregates in intraneuronal inclusions called Lewy bodies in Parkinson's disease (PD). It is composed of an N-terminal domain with a propensity to bind lipids and a C-terminal domain rich in acidic residues (the acidic tail). The objective of this study was to examine the effect of Ca(2+) on the acidic tail conformation in lipid-bound alpha-syn. We exploit the extreme sensitivity of the band III fluorescence emission peak of the pyrene fluorophore to the polarity of its microenvironment to monitor subtle conformational response of the alpha-syn acidic tail to Ca(2+). Using recombinant human alpha-syn bearing a pyrene to probe either the N-terminal domain or the acidic tail, we noted that lipid binding resulted in an increase in band III emission intensity in the pyrene probe tagging the N-terminal domain but not that in the acidic tail. This suggests that the protein is anchored to the lipid surface via the N-terminal domain. However, addition of Ca(2+) caused an increase in band III emission intensity in the pyrene tagging the acidic tail, with a corresponding increased susceptibility to quenching by quenchers located in the lipid milieu, indicative of lipid interaction of this domain. Taken together with the increased beta-sheet content of membrane-associated alpha-syn in the presence of Ca(2+), we propose a model wherein initial lipid interaction occurs via the N-terminal domain, followed by a Ca(2+)-triggered membrane association of the acidic tail as a potential mechanism leading to alpha-syn aggregation. These observations have direct implications in the role of age-related oxidative stress and the attendant cellular Ca(2+) dysregulation as critical factors in alpha-syn aggregation in PD.     

Configurations of the N-terminal amphipathic domain of the membrane-bound M13 major coat protein

The M13 major coat protein has been extensively studied in detergent-based and phospholipid model systems to elucidate its structure. This resulted in an L-shaped model structure of the protein in membranes. An amphipathic alpha-helical N-terminal arm, which is parallel to the surface of the membrane, is connected via a flexible linker to an alpha-helical transmembrane domain. In the present study, a fluorescence polarity probe or ESR spin probe is attached to the SH group of a series of N-terminal single cysteine mutants, which were reconstituted into DOPC model membranes. With ESR spectroscopy, we measured the local mobility of N-terminal positions of the protein in the membrane. This is supplemented with relative depth measurements at these positions by fluorescence spectroscopy via the wavelength of maximum emission and fluorescence quenching. Results show the existence of at least two possible configurations of the M13 amphipathic N-terminal arm on the ESR time scale. The arm is bound either to the membrane surface or in the water phase. The removal or addition of a hydrophobic membrane-anchor by site-specific mutagenesis changes the ratio between the membrane-bound and the water phase fraction.     

The use of n-(9-anthroyloxy) fatty acids to determine fluidity and polarity gradients in phospholipid bilayers

A set of n-(9-anthroyloxy) fatty acid probes (n = 2, 6, 9, 12) have been used to examine gradients in fluorescence polarization, lifetime (tau F), relative quantum yield (phi rel) and positions of emission maxima (lambda max) through bilayers composed of synthetic phospholipids. The fluorophores of these probes report the environment at a graded series of depths from the surface to the centre of the bilayer structure. 1. Polarizations decrease as the fluorophore is moved deeper into the bilayer indicating greater rotational motion of the fluorophore in the hydrocarbon core of the bilayer. 2. The different responses of the probe diphenylhexatriene and the anthroyloxy fatty acids to the action of cholesterol on lipid bilayers are discussed in terms of the orientation of these probes in the bilayer and the types of anisotropic rotational motions which result in depolarization of fluorescence. 3. Stearic acid derivatives which have the fluorophore in the 6-, 9- and 12-positions along the acyl chain have a similar response to solvent polarity as measured by values of lambda max and phi rel in a variety of organic solvents. 4. The position of the emission maximum has little dependence on solvent viscosity, but viscosity does change the degree of vibrational structure seen in the emission spectrum. The vibrational structure itself may be used as an indication of the 'mciroviscosity' gradient in the transverse plane of the bilayer. 5. Values of lambda max, tau F and phi rel indicate that a gradient of polarity exists from the surface to the centre of the bilayer. For dipalmitoyl phosphatidylcholine in the crystalline phase, cholesterol acts to make this polarity gradient shallower.     

Interaction between carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and saturating concentrations of Calcofluor White. A fluorescence study

Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously followed by fluorescence titration of the Trp residues of the protein. A stoichiometry of one Calcofluor for one protein has been found [J.R. Albani and Y.D. Plancke, Carbohydr. Res., 318 (1999) 193-200]. Alpha1-acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. Since binding of Calcofluor to alpha1-acid glycoprotein occurs mainly on the carbohydrate residues, we studied in the present work the interaction between Calcofluor and the protein by following the fluorescence change of the fluorophore. In order to establish the role of the sialic acid residues in the interaction, the experiments were performed with the sialylated and asialylated protein. Interaction of Calcofluor with sialylated alpha1-acid glycoprotein induces a red shift of the emission maximum of the fluorophore from 438 to 450 nm at saturation (one Calcofluor for one sialic acid) and an increase in the fluorescence intensity. At saturation the fluorescence intensity increase levels off. Binding of Calcofluor to asialylated acid glycoprotein does not change the position of the emission maximum of the fluorophore and induces a decrease in its fluorescence intensity. Saturation occurs when 10 molecules of Calcofluor are bound to 1 mol of alpha1-acid glycoprotein. Since the protein contains five heteropolysaccharide groups, we have 2 mol of Calcofluor for each group. Addition of free sialic acid to Calcofluor induces a continuous decrease in the fluorescence intensity of the fluorophore but does not change the position of the emission maximum. Our results confirm the presence of a defined spatial conformation of the sialic acid residues, a conformation that disappears when they are free in solution. Dynamics studies on Calcofluor White and the carbohydrate residues of alpha1-acid glycoprotein are also performed at saturating concentrations of Calcofluor using the red-edge excitation spectra and steady-state anisotropy studies. The red-edge excitation spectra experiments show an important shift (13 nm) of the fluorescence emission maximum of the probe. This reveals that emission of Calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that the microenvironment of bound Calcofluor is rigid, inducing in this way the rigidity of the fluorophore itself, a result confirmed by anisotropy studies.     

The Spectral Properties of (-)-Epigallocatechin 3-O-Gallate (EGCG) Fluorescence in Different Solvents: Dependence on Solvent Polarity

(-)-Epigallocatechin 3-O-gallate (EGCG) a molecule found in green tea and known for a plethora of bioactive properties is an inhibitor of heat shock protein 90 (HSP90), a protein of interest as a target for cancer and neuroprotection. Determination of the spectral properties of EGCG fluorescence in environments similar to those of binding sites found in proteins provides an important tool to directly study protein-EGCG interactions. The goal of this study is to examine the spectral properties of EGCG fluorescence in an aqueous buffer (AB) at pH=7.0, acetonitrile (AN) (a polar aprotic solvent), dimethylsulfoxide (DMSO) (a polar aprotic solvent), and ethanol (EtOH) (a polar protic solvent). We demonstrate that EGCG is a highly fluorescent molecule when excited at approximately 275 nm with emission maxima between 350 and 400 nm depending on solvent. Another smaller excitation peak was found when EGCG is excited at approximately 235 nm with maximum emission between 340 and 400 nm. We found that the fluorescence intensity (FI) of EGCG in AB at pH=7.0 is significantly quenched, and that it is about 85 times higher in an aprotic solvent DMSO. The Stokes shifts of EGCG fluorescence were determined by solvent polarity. In addition, while the emission maxima of EGCG fluorescence in AB, DMSO, and EtOH follow the Lippert-Mataga equation, its fluorescence in AN points to non-specific solvent effects on EGCG fluorescence. We conclude that significant solvent-dependent changes in both fluorescence intensity and fluorescence emission shifts can be effectively used to distinguish EGCG in aqueous solutions from EGCG in environments of different polarity, and, thus, can be used to study specific EGCG binding to protein binding sites where the environment is often different from aqueous in terms of polarity.     

Probing pH and pressure effects on the apomyoglobin heme pocket with the 2''-(N,N-dimethylamino)-6-naphthoyl-4-trans-cyclohexanoic acid fluorophore.

The environmentally sensitive fluorophore 2'-(N,N-dimethylamino)-6-naphthoyl-4-trans-cyclohexanoic acid (DANCA) has been used to probe the apomyoglobin heme pocket. The unexpected polarity of this domain is generally interpreted as arising from dynamic dipolar relaxation of the peptide dipoles surrounding the heme pocket. In the present work we reexamine the photophysical properties of DANCA in a variety of solvents and complexed with apomyoglobin (apoMb) to further probe the heme pocket environment as a function of external solvent conditions. Absorption and excitation spectra in a number of solvents are consistent with the well-known pi*<--pi (LE) and pi*<--n (CT) electronic absorption transitions observed for naphthylamine derivatives. Dual emission is also a well-documented property of such derivatives. Based on the time scale of the heterogeneity in the decay of the DANCA fluorophore observed in a series of solvents, we propose that the emission properties of DANCA in apoMb are not uniquely attributable to dynamic relaxation events, but also reflect dual emission from both a long-lived, red CT state and the shorter-lived, blue LE state. The pH studies in the range of pH 5-9 of the emission properties of DANCA in apoMb support this hypothesis. They also suggest a specific interaction of DANCA with one or both of the pocket histidyl residues, which leads to a drastic static quenching and red shift of the bound DANCA fluorescence upon protonation. Similar effects are observed with increasing pressure, indicating that these two perturbations alter the DANCA-apoMb complex in a similar fashion. The pressure-induced form of the protein is distinct both energetically and structurally from the previously characterized acid intermediate, in that it is populated above pH 5 and retains a significant degree of integrity of the heme pocket.     

Dynamics of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and emission anisotropy studies of Calcofluor White

Dynamics studies on Calcofluor White bound to the carbohydrate residues of sialylated and asialylated alpha 1-acid glycoprotein (orosomucoid) have been performed. The interaction between the fluorophore and the protein was found to occur preferentially with the glycan residues with a dependence on their spatial conformation. In the presence of sialylated alpha 1-acid glycoprotein, excitation at the red edge of the absorption spectrum of calcofluor does not lead to a shift in the fluorescence emission maximum (440 nm) of the fluorophore. Thus, the emission of calcofluor occurs from a relaxed state. This is confirmed by anisotropy studies as a function of temperature (Perrin plot). In the presence of asialylated alpha 1-acid glycoprotein, red-edge excitation spectra show an important shift (8 nm) of the fluorescence emission maximum of the probe. This reveals that emission of calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that Calcofluor molecules are bound tightly to the carbohydrate residues, a result confirmed by anisotropy studies.     

The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue

Hydrophobic sites on the surface of protein molecules are thought to have important functional roles. The identification of such sites can provide information about the function and mode of interaction with other cellular components. While the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, strong intrinsic quenching of Nile red and ANSA dye fluorescence is observed on binding to a cytochrome c('). Fluorescence quenching is also observed to take place in the presence of a variety of other biologically important molecules which can compromise the quantitative determination of binding constants. Absorption difference spectroscopy is shown not to be sensitive to the presence of fluorescence quenchers but sensitive enough to measure binding constants. The dye BPB is shown to bind to the same hydrophobic sites on proteins as polarity-sensitive fluorescence probes. The absorption spectrum of BPB is also observed to be polarity sensitive. A binding constant of 3x10(6)M(-1) for BPB to BSA has been measured by absorption difference spectroscopy. An empirical correlation is observed between the shape of the absorption difference spectrum of BPB and the polarity of the environment. The results indicate that absorption difference spectroscopy of BPB provides a valuable supplement to fluorescence for determining the presence of hydrophobic sites on the surface of proteins as well as a method for measuring binding constants.     

Development of a push–π–pull phenothiazine-vinyl-isophorone fluorophore: a novel solvatochromic and pH indicator

Knoevenagel condensation of phenothiazine-3,7-dicarbaldehyde with an isophorone yielded a new phenothiazine derivative ( PTZ-c ) fluorophore. The solvatochromic and pH-sensing abilities of PTZ-c , an asymmetric fluorophore with a single isophorone molecule, were shown to be exceptional. PTZ-c produced very delicate absorbance and emission spectra. When the polarity of the solvent was increased, the PTZ-c emission spectra showed greater sensitivity than the absorption spectra. Multiple spectroscopic techniques, including Fourier transform infrared spectroscopy, nuclear magnetic resonance, and mass spectrometry, were used to characterize the manufactured PTZ-c sensor. To demonstrate the beneficial solvatochromic behaviour associated with intramolecular charge transfer, the absorption spectra of the synthesized DA PTZ-c dye were analyzed in different solvents of varying polarity. Band intensity and the wavelength of PTZ-c emission were also found to be highly solvent dependent. It was observed that when solvent polarity was increased to a maximum of 4122 cm⁻¹, Stokes’ shift also increased. To analyze the Stokes' shift that depended on the solvent, a linear correlation between solvation and energy was used. An investigation of PTZ-c quantum yield (ф) was also conducted. Both the absorbance and fluorescence spectra of the sensor in dimethylformamide as a function of pH were studied. A fluorescence peak was seen at 562 nm, whereas the greatest absorption wavelengths were found at 403 and 317 nm. It was shown that the pH-sensing mechanism depended on protons removed from the PTZ-c chromophore, which caused a colour shift and variation in both emission and colorimetric properties.     

New BODIPY lipid probes for fluorescence studies of membranes

Many fluorescent lipid probes tend to loop back to the membrane interface when attached to a lipid acyl chain rather than embedding deeply into the bilayer. To achieve maximum embedding of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore into the bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholines was synthesized bearing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me(4)-BODIPY-8) at the end of C(3)-, C(5)-, C(7)-, or C(9)-acyl. A strategy was used of symmetrically dispersing the methyl groups at BODIPY ring positions 1, 3, 5, and 7 to decrease fluorophore polarity. Iodide quenching of the phosphatidylcholine probes in bilayer vesicles confirmed that the Me(4)-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of Me(4)-BODIPY-8 fluorescence quenching by phosphatidylcholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of Me(4)-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation of <10 mol% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed the retention of many features of other BODIPY derivatives (i.e., absorption and emission wavelength maxima near 498 nm and approximately 506-515 nm) but also showed the absence of the 620-630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me(4)-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.     

Exploring structural change of protein bovine serum albumin by external perturbation using extrinsic fluorescence probe: spectroscopic measurement, molecular docking and molecular dynamics simulation

Structural modification through binding interaction of plasma protein bovine serum albumin (BSA) with an extrinsic charge transfer fluorophore 5-(4-dimethylamino-phenyl)-penta-2,4-dienoic acid (DMAPPDA) and its response to external perturbation due to interactions with surfactant sodium dodecyl sulphate (SDS) have been explored at physiological pH by steady state absorption, emission, fluorescence anisotropy, red edge excitation shift, far-UV circular dichroism and time resolved spectral measurements in combination with Molecular Docking and Molecular Dynamics (MD) simulation. Interaction of the probe with BSA is reflected by a small change in protein secondary structure with fluorescence enhancement and blue shift of probe emission. Molecular docking studies revealed that the probe binds to the hydrophobic cavity of sub-domain IIA of BSA. The distance for energy transfer from the tryptophan of BSA to the bound DMAPPDA measured by Fluorescence Resonance Energy Transfer is in good agreement with the molecular docking results. MD simulation predicts stabilization of the complex with respect to the bare molecule. Interaction of BSA and SDS with DMAPPDA supports the movement of the probe from hydrophilic free water region to a more restricted hydrophobic zone inside the protein.     

肽链及蛋白质N-末端羰酰荧光衍生物的形成

短肽及胰岛素的N-末端经Dixpn转氨后具有荧光发色性质,其激发与发射波长随肽链氨基酸残基的种类和数量的不同而有差异,其范围大约为Ex 312—333nm;Em 398—408nm;量子产率为0.020—0.03.荧光发色团的基本化学结构可能是N-末端α-羰酰基及其相连的酰胺基,并且N-末端第二及第三位氨基酸残基对其荧光性质有影响.在碱性溶液中(pH>9.0)它的荧光强度降低,但在NaCl溶液中随盐浓度的增加而增强.在不同浓度的CuHCl溶液中,羰酰三肽的荧光强度变化随其氨基酸残基的种类和构型的不同而有差异.以上结果提示;α-羰酰荧光衍生物可能作为蛋白及肽N-末端的荧光探剂,可能成为研究蛋白和肽结构与功能的一种手段.     

Fluorescence properties of methyl 8-(2-anthroyl) octanoate, a solvatochromic lipophilic probe.

Fluorescence excitation and emission spectra, relative fluorescence quantum yield phi r and fluorescence lifetime tau of methyl 8-(2-anthroyl)-octanoate have been studied in a set of organic solvents covering a large scale of polarity and in the presence of water. In this probe, the 2-anthroyl chromophore exhibits quite remarkable and unique fluorescence properties. Thus, when going from n-hexane to methanol, the maximum emission wavelength lambda em max shifts from 404 nm to 492 nm while phi r and tau increase from 1 to 17.7 and from 0.91 ns to 13.5 ns, respectively. These increments are still more accentuated in the presence of water with estimated values of 526 nm for lambda em max, 27 for phi r and 20 ns for tau in this solvent. Because of the presence of a keto group which is a hydrogen bond acceptor and which can conjugate with the aromatic ring so as to provide the chromophore with a high dipole moment, the fluorescence properties of the probe strongly depend on the polarity of the surrounding medium. They can be accounted for in terms of general solvent effects (dipolar solute/solvent interactions) in the presence of aprotic solvents and in terms of specific solvent effects (hydrogen bonding) in protic solvents. Such properties of solvatochromism make the 2-anthroyl chromophore, after 8-(2-anthroyl)octanoic acid has been attached to phospholipids (E. Perochon and J.F. Tocanne (1991) Chem. Phys. Lipids 58, 7-17) a potential tool for studying microenvironmental polarity in biological membranes.     

Polarity of lipid bilayers. A fluorescence investigation.

Through steady-state and time-resolved fluorescence experiments, the polarity of the bilayers of egg phosphatidylcholine vesicles was studied by means of the solvatochromic 2-anthroyl fluorophore which we have recently introduced for investigating the environmental micropolarity of membranes and which was incorporated synthetically in phosphatidylcholine molecules (anthroyl-PC) in the form of 8-(2-anthroyl)octanoic acid. Fluorescence quenching experiments carried out with N,N-dimethylaniline and 12-doxylstearic acid as quenchers showed that the 2-anthroyl chromophore was located in depth in the hydrophobic region of the lipid bilayer corresponding to the C9-C16 segment of the acyl chains. Steady-state fluorescence spectroscopy revealed a nonstructured and red-shifted (lambda em(max) = 464 nm) spectrum for the probe in egg-PC bilayers, which greatly differed from the structured and blue (lambda em(max) = 404 nm) spectrum the fluorophore was shown to display in n-hexane. While the fluorescence decays of the fluorophore in organic solvents were monoexponential, three exponentials were required to account for the fluorescence decays of anthroyl-PC in egg-PC vesicles, with average characteristic times of 1.5 ns, 5.5 ns, and 20 ns. These lifetime values were independent of the emission wavelength used. Addition of cholesterol to the lipid did not alter these tau values. One just observed an increase in the fractional population of the 1.5-ns short-living species detrimental to the population of the 20-ns long-living ones. These observations enabled time-resolved fluorescence spectroscopy measurements to be achieved in the case of the 1/1 (mol/mol) egg-PC/cholesterol mixture. Three distinct decay associated spectra (DAS) were recorded, with maximum emission wavelengths, respectively, of 410 nm, 440 nm, and 477 nm for the 1.5-ns, 6-ns, and 20-ns lifetimes found in this system. On account of the properties and the polarity scale previously established for the 2-anthroyl chromophore in organic solvents, these data strongly suggest the occurrence of three distinct excited states for anthroyl-PC in egg-PC bilayers, corresponding to three environments for the 2-anthroyl chromophore, differing in polarity. The lifetime of 1.5 ns and the corresponding structured and blue (lambda em(max) = 410 nm) DAS account for a hydrophobic environment, with an apparent dielectric constant of 2, which is that expected for the hydrophobic core of the lipid bilayer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membrane assembly of the bacteriophage Pf3 major coat protein

The Pf3 major coat protein of the Pf3 bacteriophage is stored in the inner membrane of the infected cell during the reproductive cycle. The protein consists of 44 amino acids, and contains an acidic amphipathic N-terminal domain, a hydrophobic domain, and a short basic C-terminal domain. The mainly alpha-helical membrane-bound protein traverses the membrane once, leaving the C-terminus in the cytoplasm and the N-terminus in the periplasm. A cysteine-scanning approach was followed to measure which part of the membrane-bound Pf3 protein is inside or outside the membrane. In this approach, the fluorescence probe N-[(iodoacetyl)amino]ethyl-1-sulfonaphthylamine (IAEDANS) was attached to single-cysteine mutants of the Pf3 coat protein. The labeled mutant coat proteins were reconstituted into the phospholipid DOPC/DOPG (80/20 molar ratio) and DOPE/DOPG (80/20 molar ratio) model membranes. We subsequently studied the fluorescence characteristics at the different positions in the protein. We measured the local polarity of the environment of the probe, as well as the accessibility of the probe to the fluorescence quencher acrylamide. The results of this study show a single membrane-spanning protein with both the C- and N-termini remaining close to the surface of the membrane. A nearly identical result was seen previously for the membrane-bound M13 coat protein. On the basis of a comparison between the results from both studies, we suggest an "L-shaped" membrane-bound model for the Pf3 coat protein. DOPE-containing model membranes revealed a higher polarity, and quenching efficiency at the membrane/water interface. Furthermore, from the outside to the inside of the membrane, a steeper polarity gradient was measured at the PE/PG interface as compared to the PC/PG interface. These results suggest a thinner interface for DOPE/DOPG than for DOPC/DOPG membranes.     

Styryl-BODIPY based red-emitting fluorescent OFF-ON molecular probe for specific detection of cysteine

We have synthesized a styryl boron-dipyrromethene (BODIPY)/2,4-dinitrobenzenesulfonyl (DNBS) dyad based red-emitting molecular probe for specific detection of cysteine among the biological thiols. The probe shows intensive absorption at 556 nm and the probe is non-fluorescent. The DNBS moiety can be cleaved off by thiols, the red emission of the BODIPY fluorophore at 590 nm is switched on, with an emission enhancement of 46-fold. The probe shows good specificity toward cysteine over other biological molecules, such as glutathione and amino acids. The emission of the probe is pH-independent in the physiological pH range. The probe is used for fluorescent imaging of cellular thiols. Theoretical calculations based on density functional theory (DFT) were used to elucidate the fluorescence sensing mechanism of the probe, which indicate a dark excited state (S(1)) for the probe but an emissive excited state (S(1)) for the cleaved product (i.e. the fluorophore).