Thermal denaturation and fluorescence study of nucleosomes containing non-histone chromosomal protein HMG2

Interaction of calf thymus non-histone chromosomal protein HMG2 with H1,H5-depleted nucleosomes from chicken erythrocytes was studied by means of thermal denaturation and an N-(3-pyrene)maleimide fluorescence probe. Under low ionic conditions (2 mM Tris buffer plus EDTA) addition of 1-2 molecules of HMG2 per nucleosome markedly stabilized the segment of the linker DNA against thermal denaturation. Under approximately physiological ionic conditions (0.1 M NaCl) addition of two HMG2 molecules per nucleosome, labeled by N-(3-pyrene)maleimide at the sulfhydryl groups of Cys-110 of histones H3, resulted in a decrease of the pyrene excimer fluorescence corresponding to the slight movement of the sulfhydryl groups of the two histone H3 molecules apart.     

Pyrene derivatives as markers of transbilayer effect of lipid peroxidation on neuronal membranes

Two different pyrene derivatives, namely 12-(1-pyrene)dodecanoic acid (P12-FA) and N-(12-(1-pyrene)dodecanoyl)-galactosylsphingosine I3-sulfate (P12-CS) have been used to follow lipid peroxidation both in model and natural membranes. The malondialdehyde (MDA) production in small unilamellar vesicles of dipalmitoylphosphatidylcholine/arachidonic acid (80:20, molar ratio), symmetrically labelled with both probes determined a progressive decrease of pyrene fluorescence due to an involvement of pyrene in the peroxidative reaction. Nervous membranes are particularly sensitive to lipid oxidation which differentially acts on the two layers of the membrane determining a greater rigidity of the exofacial one. Thus, we consider the possibility to asymmetrically introduce the pyrene ring, as P12-FA or P12-CS, in synaptosomes for monitoring lipid peroxidation in each layer of the membrane. The amount of the two probes incorporated in the membrane was 20 +/- 3 and 10 +/- 2 nmol/mg of protein for P12-FA and P12-CS, respectively. P12-FA was symmetrically distributed in the two layers, whereas 95% of P12-CS was incorporated in the exofacial layer of the membrane as determined by TNBS measurements. The decrease in fluorescence of synaptosome associated pyrene was, in the early stages of lipid peroxidation, greater for P12-CS than for P12-FA labelled membranes, indicating a greater susceptibility of the exofacial layer to iron-induced peroxidation.     

Pyrene-labeled cardiac troponin C. Effect of Ca2+ on monomer and excimer fluorescence in solution and in myofibrils.

The two cysteine residues (Cys-35 and Cys-84) of bovine cardiac troponin C (cTnC) were labeled with the pyrene-containing SH-reactive compounds, N-(1-pyrene) maleimide, and N-(1-pyrene)iodoacetamide in order to study conformational changes in the regulatory domain of cTnC associated with cation binding and cross-bridge attachment. The labeled cTnC exhibits the characteristic fluorescence spectrum of pyrene with two sharp monomer fluorescence peaks and one broad excimer fluorescence peak. The excimer fluorescence results from dimerization of adjacent pyrene groups. With metal binding (Mg2+ or Ca2+) to the high affinity sites of cTnC (sites III and IV), there is a small decrease in monomer fluorescence but no effect on excimer fluorescence. In contrast, Ca2+ binding to the low affinity regulatory (site II) site elicits an increase in monomer fluorescence and a reduction in excimer fluorescence. These results can be accounted for by assuming that the pyrene attached to Cys-84 is drawn into a hydrophobic pocket formed by the binding of Ca2+ to site II. When the labeled cTnC is incorporated into the troponin complex or substituted into cardiac myofibrils the monomer fluorescence is enhanced while the excimer fluorescence is reduced. This suggests that the association with other regulatory components in the thin filament might influence the proximity (or mobility) of the two pyrene groups in a way similar to that of Ca2+ binding. With the binding of Ca2+ to site II the excimer fluorescence is further reduced while the monomer fluorescence is not changed significantly. In myofibrils, cross-bridge detachment (5 mM MgATP, pCa 8.0) causes a reduction in monomer fluorescence but has no effect on excimer fluorescence. However, saturation of the cTnC with Ca2+ reduces excimer fluorescence but causes no further change in monomer fluorescence. Thus, the pyrene fluorescence spectra define the different conformations of cTnC associated with weak-binding, cycling, and rigor cross-bridges.     

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.

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.     

The interaction of prostaglandins with human serum lipoproteins

Using high density and low density lipoproteins (HDL and LDL) labeled with fluorescent analogues of phosphatidylcholine or sphingomyelin it was found that low amounts (10^–12 M) of prostaglandins E₁ and F₂ induced different structural rearrangements of the lipoprotein surface, whereas prostaglandins E₂ and F₁ had no effect. The effects of prostaglandin E₁ on HDL were largely paralled by those of this prostaglandin on synthetic recombinants prepared from pure apolipoprotein A₁, phospholipids and cholesterol and were demonstrated to be caused by prostaglandin-apolipoprotein interaction. The interaction resembled that of a ligand with a specific receptor protein because it was specific, reversible, concentration and temperature dependent and saturable. However the retaining capacity of HDL or LDL for prostaglandin E₁ as determined by equilibrium dialysis was very low and a single prostaglandin E₁ molecule was able to induce structural changes in large numbers of discrete lipoprotein particles. To explain this remarkable fact a non-equilibrium model of ligand-receptor interaction is proposed. According to that model in open systems characterized by weak ligand-receptor binding, high diffusion rate of the ligand and long relaxation times which exceed the interval between two successive receptor occupations, the ligand-induced changes will accumulate, resulting in transformation of the system into a new state which may be far away from equilibrium. It is emphasized that the low mobility of lipids constituting the environment of the receptor protein plays a critcal role in this type of signal amplification.It was further demonstrated that the PGE₁-induced changes of the lipoprotein surface resulted in an enhancement of LDL-to-HDL transfer of cholesterol esters and phosphatidylcholine especially in the presence of serum lipid transfer proteins. The acceleration of the interlipoprotein transfer caused by prostaglandin E₁ in turn increases the rate of cholesterol esterification in serum. It is suggested that in such a way prostaglandin E₁ may influence the homeostasis of cholesterol.Abbreviations LDL low density lioproteins - HDL high density lipoproteins - PG prostaglandin - ASM anthrylvinyl-labeled sphingomyelin (N-12-(9-anthryl)-11-trans-dodecanoylsphingosin-1-phosphocholine - APC anthrylvinylphosphatidylcholine (1-radyl-2-[(9-anthryl)-11-transdodecanoyl)-sn-glycerophosphocholine - NAP-SM nitroazidophenyl labeled sphingomyelin (N-[N-(2-nitro-4azidophenyl)-12-aminododecanoyl]-sphingosin-1-phosphocholine) - NAP-PC adizophenyl labeled phosphatidylcholine (1-radyl-2-[N-(2-nitro-4azidophenyl)-12-aminododecanoyl]-sn-glycero-3-phosphocholine - DPPC dipalmitoylphosphatidylcholine - P fluorescence polarization - E parameter of tryptophanyl to ASM resonance energy transfer - LEP lipid-exchange protein     

Ca2+-induced conformational changes in cardiac troponin C as measured by N-(1-pyrene)maleimide fluorescence

Bovine cardiac troponin C was modified by N-(1-pyrene)maleimide at Cys-35 and Cys-84; the Ca2+-induced conformational changes were followed by measuring pyrene fluorescence. In isolated troponin C, the saturation of Ca2+, Mg2+-sites leads to a simultaneous increase in the pyrene monomer as well as to a decrease in the pyrene excimer fluorescence, whereas the saturation of Ca2+-specific sites results in a slight decrease in the fluorescence of pyrene monomer. Troponin T does not influence the dependence of pyrene-troponin C fluorescence on Ca2+ concentration. Within the equimolar complex of troponin C and troponin I, the saturation of Ca2+, Mg2+-sites has no effect on pyrene fluorescence, whereas the saturation of Ca2+-specific sites leads to a simultaneous decrease of both pyrene monomer and pyrene excimer fluorescence. It is supposed that troponin I diminishes the conformational changes in troponin C that are induced by the saturation of Ca2+, Mg2+-sites and enhances the conformational changes induced by the saturation of Ca2+-specific sites of troponin C.     

Synthesis of novel bispyrene diamines and their application as ratiometric fluorescent probes for detection of DNA

Fluorescent DNA probes with 1,6-hexanediyl as the linker between two pyrenes, phenylpyrenes or phenylethynyl pyrene fluorophores were synthesized (Py-1, Py-2 and Py-3) and their interactions with DNA were studied by UV–vis absorption spectra, fluorescence spectra and viscosity measurements. The probes show red-shifted emission compared with pyrene (up to 20 nm). We found the interaction of these probes with DNA can be either intercalation or groove binding. Ratiometric fluorometry (ratio of the monomer and excimer emission intensity versus concentration of DNA) was achieved with these probes for DNA quantification (with limit of detection, LOD, up to 0.1 μg/mL). We also found that the undesired oxygen sensitivity of the emission intensity of pyrene fluorophore can be greatly suppressed by extending the π-conjugation framework of pyrene (the I_Ar/I_air value is decreased from 8.10 for pyrene to less than 2.20 for the DNA probes described herein).     

Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.Abbreviations dipyr₄ sn-1,2-(pyrenylbutyl) - dipyr₁₀ sn-1,2-(pyrenyldecanoyl). - DMPC dimyristoyl-phosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - PC phosphatidylcholine - PS phosphatidylserine Correspondence to: A. J. W. G. Visser     

Monomer and excimer fluorescence of horse plasma gelsolin labelled with N-(1-pyrenyl)iodoacetamide.

Horse plasma gelsolin was labelled with the sulfhydryl-specific fluorescent reagent N-(1-pyrenyl)iodoacetamide. The level of incorporation of probe was 1.6 +/- 0.3 mol pyrene/mol gelsolin. The circular dichroism spectrum of pyrenyl-gelsolin and its ability to interact with muscle actin were not different from that found for unmodified gelsolin. The emission from pyrenyl-gelsolin was dominated by a broad emission band centred near 483 nm, characteristic of the presence of pyrene excimers. Analysis of excitation spectra for the monomer and excimer-type fluorescence suggested that ground-state interactions may occur between adjacent pyrenes in the gelsolin structure. In the case either of excimer formation or of ground-state pyrene-pyrene interactions in doubly labelled gelsolin molecules, the modified Cys residues must be in close proximity in the folded protein structure. Thermal denaturation of gelsolin could be monitored by observing the decrease in excimer emission that accompanied heating and unfolding of the tertiary structure. While heat treatment alone did not eliminate excimer fluorescence, digestion of gelsolin with chymotrypsin completely abolished such emission. Also, pyrenyl-gelsolin prepared and studied in 6 M guanidine-HCl exhibited fluorescence characteristic of pyrene monomers exclusively.     

Fluorescence decay of pyrene in small and large unilamellar L,α-Dipalmitoylphosphatidylcholine vesicles above and below the phase transition temperature

The fluorescence decays of pyrene in small and large unilamellar L,-dipalmitoylphosphatidylcholine vesicles have been investigated as a function of probe concentration and temperature. When the molar ratio of pyrene to phospholipid equals 1:3000, no excimer emission is observed and the fluorescence decays are mono-exponential. When this ratio is equal to or higher than 1:120, excimer formation is observed.Above the phase transition temperature the observed fluorescence decays of monomer and excimer can be adequately described by a bi-exponential function. The monomer decays can be equally well fitted to a decay law which takes into account a time-dependence in the probe diffusion rate constant. The fluorescence decay kinetics are compatible with the excimer formation scheme which is valid in an isotropic medium. The excimer lifetime and the (apparent) rate constant of excimer formation have been determined as a function of probe concentration at different temperatures above the phase transition temperature. The activation energy of excimer formation is found to be 29.4±1.3 kJ/mol. In small unilamellar vesicles the diffusion constant associated with the pyrene excimer formation process varies from 8.0x10^-7 cm²/s at 40°C to 2.2x10^-6 cm²/s at 70°C.Below the phase transition temperature the monomer decays can be described by a decay law which takes into account a time dependence of the rate constant of excimer formation. The lateral diffusion coefficient of pyrene calculated from the decay fitting parameters of the monomer region varies from 4.0x10^-9 cm²/s at 20°C to 7.9x10^-8 cm²/s at 35°C. No significant difference could be observed between the pyrene fluorescence decay kinetics in small and large unilamellar vesicles.Abbreviations SUV small unilamellar vesicles - LUV large unilamellar vesicles - DPPC dipalmitoylphosphatidylcholine - DMPC dimyristoylphosphatidylcholine - FRAP fluorescence recovery after photobleaching Part of this research has been presented at the 5th international symposium on surfactants in solution. Bordeaux, July 9th–13th 1984     

Pyrene-labeled gangliosides: micelle formation in aqueous solution, lateral diffusion, and thermotropic behavior in phosphatidylcholine bilayers

By use of the excimer technique, the formation in aqueous solution of pyrene-labeled ganglioside micelles and their lateral diffusion and distribution in phosphatidylcholine membranes were investigated. For these studies 12-(1-pyrenyl)dodecanoic acid was covalently attached to the ceramide part of lysogangliosides GM1, GM2, GM3, GD1a, and GD1b. The 12-(1-pyrenyl)dodecanoic acid substitute of phosphatidylcholine was used for comparison. All pyrene-labeled gangliosides were present in aqueous solution in a predominantly micellar form down to 2 X 10(-8) M, which is the technical limit of this method. The tendency to aggregate is highest for PyGD1a and PyGD1b. In fluid dipalmitoylphosphatidylcholine bilayers the excimer-to-monomer fluorescence intensity ratio of pyrene-labeled gangliosides PyGM1, PyGM2, PyGM3, PyGD1a, and PyGD1b increases linearly with ganglioside concentration. The calculated diffusion coefficients for gangliosides are comparable to 1.6 X 10(-7) cm2/s, which is the diffusion coefficient of pyrene-labeled phosphatidylcholine [Galla, H.-J., & Hartmann, W. (1980) Chem. Phys. Lipids 27, 199-219]. In comparison to phosphatidylcholine, the diffusion of monosialogangliosides is slightly increased, with that diffusion of disialogangliosides being slightly decreased. Ca2+ ions up to 200 mM do not affect ganglioside diffusion significantly. The shape of the lipid phase transition curves obtained by the excimer technique yields information on the lateral distribution of the tested probe molecules. Pyrene-labeled phosphatidylcholine was taken as reference for a system with complete miscibility but nonideal mixing. 1-Acyl-2-[10-(1-pyrenyl)decanoyl]-sn-glycero-3-phosphocholine (PyPC) is known to be randomly distributed in the gel and in the fluid-crystalline lipid phase of dipalmitoylphosphatidylcholine bilayer membranes. It distributes preferentially into the fluid phase in the phase-transition region. In comparison, PyPC in dimyristoylphosphatidylcholine membranes is an example of a system with nearly ideal mixing [Hresko, R. C., Sugar, J. P., Barenholz, Y., & Thompson, T. E. (1986) Biochemistry 25, 3813-3828]. Phase-transition curves of pyrene-labeled gangliosides exemplify a nearly ideal mixing system with PyGD1a or PyGD1b producing best effects. The monosialogangliosides, however, exhibit less ideality of mixing, the deviation from an ideal mixing behavior increasing with decreasing number of both neutral sugar residues and sialic acid groups. Addition of Ca2+ triggers a tightening of the phosphatidylcholine bilayer and thus induces a change in the lateral distribution of the gangliosides at the phase transition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Asymmetric structural features in single supported lipid bilayers containing cholesterol and GM1 resolved with synchrotron X-Ray reflectivity

The cell membrane comprises numerous protein and lipid molecules capable of asymmetric organization between leaflets and liquid-liquid phase separation. We use single supported lipid bilayers (SLBs) to model cell membranes, and study how cholesterol and asymmetrically oriented ganglioside receptor G_M1 affect membrane structure using synchrotron x-ray reflectivity. Using mixtures of cholesterol, sphingomyelin, and 1,2-dioleoyl-sn-glycero-3-phosphocholine, we characterize the structure of liquid-ordered and liquid-disordered SLBs in terms of acyl-chain density, headgroup size, and leaflet thickness. SLBs modeling the liquid-ordered phase are 10 Å thicker and have a higher acyl-chain electron density (〈ρ_chain〉 = 0.33 e⁻/ų) compared to SLBs modeling the liquid-disordered phase, or pure phosphatidylcholine SLBs (〈ρ_chain〉 = 0.28 e⁻/ų). Incorporating G_M1 into the distal bilayer leaflet results in membrane asymmetry and thickening of the leaflet of 4-9 Å. The structural effect of G_M1 is more complex in SLBs of cholesterol/sphingomyelin/1,2-dioleoyl-sn-glycero-3-phosphocholine, where the distal chains show a high electron density (〈ρ_chain〉 = 0.33 e⁻/ų) and the lipid diffusion constant is reduced by ∼50%, as measured by fluorescence microscopy. These results give quantitative information about the leaflet asymmetry and electron density changes induced by receptor molecules that penetrate a single lipid bilayer.     

Excimer fluorescence of pyrene-tropomyosin adducts

Studies of the fluorescence of N-(1-pyrene)maleimide and N-(1-pyrenyl)iodoacetamide with actin from rabbit skeletal muscle tropomyosin revealed the presence of excimer fluorescence characterized by a broad emission band at 480 nm with a shoulder at 505 nm. Monomer fluorescence decay exhibited different lifetimes, viz., about 3, 22 and 87 ns for the pyrenemaleimide adduct; about 2.5, 11 and 51 ns for the aminolyzed maleimide adduct: and about 2.5, 15 and 74 ns for the pyrenyliodoacetamide adduct. Almost identical excimer fluorescence lifetimes were found for all adducts; about 9, 35 and 65 ns. Excimer fluorescence was sensitive to changes in ionic strength and pH of the medium while monomer fluorescence did not change. The protein denaturants guanidine hydrochloride and urea caused dissociation of the two tropomyosin subunits and partial disappearance of excimer fluorescence, but not as effectively as the hydrophobic surfactant sodium dodecyl sulfate. The sensitivity of excimer fluorescence to changes in the micro-environment make these pyrene derivatives very useful probes for studying conformational changes and binding interaction of tropomyosin with other contractile proteins. The unique location of the excimer probe at tropomyosin Cys-190 and its characteristic long lifetimes could make it useful in time-resolved anisotropy studies and fluorescence energy-transfer experiments.     

Pyrene excimer fluorescence: a spatially sensitive probe to monitor lipid-induced helical rearrangement of apolipophorin III

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.     

Proximity of sulfhydryl groups in lens proteins. Excimer fluorescence of pyrene-labeled crystallins

Lens proteins labeled with the -SH-specific reagents N-(1-pyrene)-maleimide (PM) and N-(1-pyrene)-iodo-acetamide (PIA) exhibited pyrene excimer fluorescence around 480 nm. Among the gamma-fractions, only gamma II showed excimer band at room temperature with both probes PM and PIA. As the temperature increased, PM-labeled gamma IIIA, gamma IIIB, and gamma IV also began to exhibit excimer around 55 degrees C, which did not disappear at a very high temperature (85 degrees C). With PIA, gamma IIIA and gamma IVA did not show excimer at any temperature. The beta-crystallins, on the other hand, revealed a very strong excimer/monomer intensity ratio at room temperature, which decreased with an increase in temperature. Life-time measurements indicated a difference in the micro-environments around the labeled -SH residues. The origin of the excimer band as well as temperature effects on this band have been explained on the basis of intra- and inter-molecular interaction among the Cys residues in the lens proteins. The temperature-dependent CD studies further indicated retention of thermodynamic stability of the crystallins after chemical modifications. Both PM and PIA could be used conveniently to probe -SH proximity, determine the ease and extent of disulfide formation, and monitor the dynamics of lens protein conformation, all of which are critically important with regard to cataract formation.     

Organization and dynamics in micellar structural transition monitored by pyrene fluorescence

Structural transitions involving shape changes play an important role in cellular physiology. Such transition can be induced in charged micelles at a given temperature by increasing ionic strength of the medium. We have monitored the change in organization and dynamics associated with sphere-to-rod transition of SDS micelles utilizing pyrene fluorescence. We report here, utilizing changes in the ratio of pyrene vibronic peak intensities (I₁/I₃), the apparent dielectric constant experienced by pyrene in spherical SDS micelles (in absence of salt) to be ∼32. Interestingly, the apparent micellar dielectric constant exhibits a reduction with increasing NaCl concentration. The dielectric constant in rod-shaped micelles of SDS (in presence of 0.5 M NaCl) turns out to be ∼22. To the best of our knowledge, these results constitute one of the early reports on polarity estimates in rod-shaped micelles. In addition, pyrene excimer/monomer ratio shows increase in SDS micelles with increasing NaCl concentration. We interpret this increase due to an increase in average number of pyrene molecules per micelle associated with the sphere-to-rod structural transition. These results could be significant in micellar drug solubilization and delivery, and in membrane morphology changes.     

Nucleosome conformation: pH and organic solvent effects.

Monomer nucleosomes (nu 1) from chicken erythrocyte nuclei were examined in aqueous buffers (8 greater than pH greater than 3) and in solvent mixtures (i.e., water and ethanol, ethylene glycol, dioxane, dimethyl sulfoxide, 2-methyl-2,4-pentanediol, polyethylene glycol, sucrose, or urea). Circular dichroism, laser Raman spectroscopy of nu 1, and the fluorescence of nu 1 labeled with N-(3-pyrene) maleimide on thiol groups of H3 histone were employed to detect conformational transitions in nu 1. The results of pH studies were as follows: 5.5 greater than pH greater than 4.8, suppression of DNA ellipticity and no change of histone alpha-helix; 4.6 greater than pH greater than 4.2 an irreversible increase in the B character of DNA, a slight loss of histone alpha-helix, and a parallel loss of pyrene excimer fluorescence; 4 greater than pH, aggregation of nu 1 and protonation of the DNA bases C and A. Results obtained in the studies of nu 1 in solvent mixtures included the following: sharp conformational transitions that variously involved an increase in the B character of DNA, a slight loss of histone alpha-helix, and a loss of pyrene excimer. Different solvents required different concentrations to effect these conformational changes.     

Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles

Small unilamellar vesicles of egg phosphatidylcholine (PC) or dimyristoylphosphatidylcholine, mixed with small unilamellar vesicles labelled with 2-(10-(1-pyrene)decanoyl)phosphatidylcholine, exhibit a constant average size and excimer to monomer (E/M) ratio for several hours when incubated at pH 3.6 at a temperature higher than the phase transition temperature (T_c) of the lipids. Addition of bovine serum albumin to this system produces a transient turbidity increase, a fast decrease in the E/M ratio, a partial loss of vesicle-entrapped [¹⁴C]sucrose and a measurable leak-in of externally added sucrose. Sepharose 4B filtration of the system demonstrates that the E/M ratio decrease is strictly paralleled by the formation of liposomes which exhibit a low E/M ratio and a hydrodynamic radius larger than that of small unilamellar vesicles. These data demonstrate that the E/M ratio decrease can be unequivocally ascribed to a vesicle-vesicle fusion process induced by serum albumin. The rate of serum-albumin induced fusion of small unilamellar vesicles is: (a) maximal at a stoichiometric ratio of approx. 2 albumins per vesicle: (b) sensitive to the nature of the lipid and; (c) not altered when human serum albumin replaces bovine serum albumin. The rate of albumin-induced fusion of dimyristoylphosphatidylcholine small unilamellar vesicles is higher below the T_c of the lipid and increases with temperature above the T_c. The formation of protein-bound aggregates with defined stoichiometries and a high local vesicle concentration, as well as changes in the local degree of hydration, are proposed to be the driving forces for the protein-induced vesicle fusion in this system.     

Interaction of gangliosides with plasma low density lipoproteins

The role of gangliosides in the reception of low density lipoproteins (LDL) was studied using as targets mouse ascites hepatoma 22a (MAH) cells which bind LDL through a specific high affinity receptor. Low density lipoprotein binding and uptake by MAH cells decreased after brief treatment of the cells with neuraminidase to partially remove surface sialic acid residues. The LDL uptake capability of the neuraminidasetreated MAH cells was fully restored after incorporation of exogeneous G_M1- and G_D1a-gangliosides into the cell surface. In contrast, free (extracellular) gangliosides inhibited LDL uptake by native MAH cells. This inhibitory effect was seen at ganglioside concentrations corresponding to the ganglioside content of serum and was most pronounced with gangliosides whose sialic acids were linked to a terminal galactose residue (G_M3, G_D1a, G_T1b) but was smaller or absent with gangliosides whose sialic acids were attached to an internal galactose (G_M1, G_M2). The binding of gangliosides to LDL was structure and concentration dependent, saturable and trypsin sensitive. The LDL-ganglioside interaction was further investigated by steady state fluorescence spectroscopy. Changes in the LDL fluorescence polarization were observed with as little as 0.01 M concentrations of the gangliosides. The magnitude and nature of the effect depended on the type of ganglioside. We conclude that the LDL surface possesses sites recognizing specific carbohydrate sequences of glycoconjugates and that changes in the cell surface concentrations of sialic acids significantly modulate the LDL uptake. It is postulated that shedding of gangliosides into the blood stream may be a factor involved in regulation of cholesterol homeostasis.Abbreviations MAH mouse ascites hepatoma 22a - LDL low density lipoprotein - ASM anthrylvinyl-labeled sphingomyelin [N-12-(9-anthryl-trans-dodecanoyl-sphingosine-1-phosphocholine] - RITC rhodamine isothiocyanate. The designation of gangliosides follows the IUPAC-IUB recommendation [1]: G_M3, II³NeuAc-LacCer, II³-N-acetylneuraminosyllactosylceramide - G_M2 II³-NeuAc-GgOse₃Cer, II³-N-acetylneuraminosylgangliotriaosylceramide - G_M1 II³-NeuAc-GgOse₄Cer, II³-N-acetylneuraminosylgangliotetraosylceramide - G_D1a, II³ IV³(NeuAc)₂-GgOse₄Cer, II³, IV³-di(N-acetylneuraminosyl)gangliotetraosylceramide - G_T1b II³(NeuAc)₂, IV³ NeuAc-GgOse₄Cer, II³-di-N-acetylneuraminosyl, IV³-N-acetylneuraminosylgangliotetraosylceramide     

Calcium-dependent protein-protein interactions induce changes in proximity relationships of Cys48 and Cys64 in chicken skeletal troponin I.

The goal of this study was to relate conformational changes in the N-terminal domain of chicken troponin I (TnI) to Ca2+ activation of the actin-myosin interaction. The two cysteine residues in this region (Cys48 and Cys64) were labeled with two sulfhydryl-reactive pyrene-containing fluorophores [N-(1-pyrene)maleimide, and N-(1-pyrene)iodoacetamide]. The labeled TnI showed a typical fluorescence spectrum: two sharp peaks of monomer fluorescence and a broad peak of excimer fluorescence arising from the formation of an excited dimer (excimer). Results obtained show that forming a binary complex of labeled TnI with skeletal TnC (sTnC) in the absence of Ca2+ decreases the excimer fluorescence, indicating a separation of the two residues. This reduction in excimer fluorescence does not occur when labeled TnI is complexed with cardiac TnC (cTnC). The latter causes only partial activation of the Ca2+-dependent myofibrillar ATPase. The binding of Ca2+ to the two N-terminal sites of sTnC causes a significant decrease in excimer fluorescence and an increase in monomer fluorescence in complexes of labeled TnI with skeletal TnC or TnC/TnT, while Ca2+ binding to site II of cTnC only causes an increase in monomer fluorescence but no change in excimer fluorescence. Thus a conformational change in the N-terminal region of TnI may be necessary for full activation of muscle contraction.