Rigidifying Effect Research Articles

Resolution of complex fluorescence spectra of lipids and nicotinic acetylcholine receptor by multivariate analysis reveals protein-mediated effects on the receptor's immediate lipid microenvironment

Analysis of fluorescent spectra from complex biological systems containing various fluorescent probes with overlapping emission bands is a challenging task. Valuable information can be extracted from the full spectra, however, by using multivariate analysis (MA) of measurements at different wavelengths. We applied MA to spectral data of purified Torpedo nicotinic acetylcholine receptor (AChR) protein reconstituted into liposomes made up of dioleoylphosphatidic acid (DOPA) and dioleoylphosphatidylcholine (DOPC) doped with two extrinsic fluorescent probes (NBD-cholesterol/pyrene-PC). Förster resonance energy transfer (FRET) was observed between the protein and pyrene-PC and between pyrene-PC and NBD-cholesterol, leading to overlapping emission bands. Partial least squares analysis was applied to fluorescence spectra of pyrene-PC in liposomes with different DOPC/DOPA ratios, generating a model that was tested by an internal validation (leave-one-out cross-validation) and was further used to predict the apparent lipid molar ratio in AChR-containing samples. The values predicted for DOPA, the lipid with the highest Tm, indicate that the protein exerts a rigidifying effect on its lipid microenvironment. A similar conclusion was reached from excimer formation of pyrene-PC, a collisional-dependent phenomenon. The excimer/monomer ratio (E/M) at different DOPC/DOPA molar ratios revealed the restricted diffusion of the probe in AChR-containing samples in comparison to pure lipid samples devoid of protein. FRET from the AChR (donor) to pyrene-PC (acceptor) as a function of temperature was found to increase with increasing temperature, suggesting a shorter distance between AChR and pyrene PC. Taken together, the results obtained by MA on complex spectra indicate that the AChR rigidifies its surrounding lipid and prefers DOPA rather than DOPC in its immediate microenvironment. PACS Codes: 32.50.+d, 33.50.Dq

Interaction between Alzheimer's Aβ(25–35) peptide and phospholipid bilayers: The role of cholesterol

There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the accumulation of β-amyloid peptides into senile plaques, one of the hallmarks of Alzheimer's disease (AD). With the aim to clarify the molecular basis of the interaction between amyloid peptides and cellular membranes, we investigated the interaction between a cytotoxic fragment of Aβ(1–42), i.e., Aβ(25–35), and phospholipid bilayer membranes. These systems were studied by Electron Paramagnetic Resonance (EPR) spectroscopy, using phospholipids spin-labeled on the acyl chain. The effect of inclusion of charged phospholipids or/and cholesterol in the bilayer composition was considered in relation to the peptide/membrane interaction. The results show that Aβ(25–35) inserts in bilayers formed by the zwitterionic phospholipid dilauroyl phosphatidylcholine (DLPC), positioning between the outer part of the hydrophobic core and the external hydrophilic layer. This process is not significantly influenced by the inclusion of the anionic phospholipid phosphatidylglycerol (DLPG) in the bilayer, indicating the peptide insertion to be driven by hydrophobic rather than electrostatic interactions. Cholesterol plays a fundamental role in regulating the peptide/membrane association, inducing a membrane transition from a fluid-disordered to a fluid-ordered phase. At low cholesterol content, in the fluid-disordered phase, the insertion of the peptide in the membrane causes a displacement of cholesterol towards the more external part of the membrane. The crowding of cholesterol enhances its rigidifying effect on this region of the bilayer. Finally, the cholesterol-rich fluid-ordered membrane looses the ability to include Aβ(25–35).

The effect of soluble surfactant on the transient motion of a buoyancy-driven bubble

The effect of soluble surfactants on the unsteady motion and deformation of a bubble rising in an otherwise quiescent liquid contained in an axisymmetric tube is computationally studied by using a finite-difference/front-tracking method. The unsteady incompressible flow equations are solved fully coupled with the evolution equations of bulk and interfacial surfactant concentrations. The surface tension is related to the interfacial surfactant concentration by a nonlinear equation of state. The nearly spherical, ellipsoidal, and dimpled ellipsoidal-cap regimes of bubble motion are examined. It is found that the surfactant generally reduces the terminal velocity of the bubble but this reduction is most pronounced in the nearly spherical regime in which the bubble behaves similar to a solid sphere and its terminal velocity approaches that of an equivalent solid sphere. Effects of the elasticity number and the bulk and interfacial Peclet numbers are examined in the spherical and ellipsoidal regimes. It is found that the surface flow and interfacial surfactant concentration profiles exhibit the formation of a stagnant cap at the trailing end of the bubble in the ellipsoidal regime at low elasticity and high interfacial Peclet numbers. Bubble deformation is first reduced due to rigidifying effect of the surfactant but is then amplified when the elasticity number exceeds a critical value due to overall reduction in the surface tension.

Comparison of paclitaxel penetration in normal and cancerous cervical model monolayer membranes

The aim of the present study was to evaluate the penetration of paclitaxel in normal as well as cancerous human cervical monolayer membranes and to compare these results with the paclitaxel penetration in a model dipalmitoylphosphatidylcholine (DPPC) monolayer. At physiologically relevant surface pressures of 30 mN/m, equilibrium drug penetration was observed in DPPC model membrane, whereas in cervical lipid model membranes exclusion of the drug and destabilization of the membrane was observed. The maximum surface pressure increment due to penetration (Δ π max) of 600 nM paclitaxel, for DPPC monolayer was found to be 3.6, 5.4 and 5.0 times higher than those for penetration in the cancerous monolayer at surface pressures 10, 20 and 30 mN/m, respectively. At initial surface pressure 10 mN/m, the maximum surface pressure increment, for 600 nM paclitaxel penetration, of normal cervical lipid membrane was double that of the cancerous cervical lipid membrane. At 30 mN/m initial surface pressure the representative IC 50 concentration of the drug produced negligible drug penetration and significant membrane destabilization in cervical lipid model membranes. The difference in penetration profile could be due to differences in composition of the model membranes. The cholesterol level in cancerous cervical membrane was 1.5-folds higher than that in the normal cervical membrane. Apart from PC, another constituent present in 20–32% in cancerous and normal membranes is sphingomyelin (SM). Introduction of 70% SM to the DPPC monolayer decreased the Δ π max from 4.7 to 1.1 mN/m, revealing the rigidifying effect of SM which was directly proportional to the amount of SM added. Modulation of fluidity of the membranes can alter the penetration of paclitaxel in biological membranes and hence its toxicity profile.

Ribosomal Synthesis of Dehydroalanine-Containing Peptides

Dehydroalanine is a nonproteinogenic amino acid, but it is a component of a wide variety of natural products with therapeutic activities. Indeed, this alpha,beta-unsaturated residue is a highly versatile building block due to its rigidifying effect on peptide backbones and its electrophilicity which allows site-specific thiol ligations of peptides with small molecules or proteins. To harness such versatility in genetically encoded, combinatorial peptide libraries, we report a simple and robust method for the ribosomal synthesis of dehydroalanine-containing peptides. Selenalysine, a selenium-containing lysine analogue, was recruited as a masked dehydroalanine equivalent. This residue is efficiently incorporated by a reconstituted Escherichia coli translation system at high fidelity and efficiency despite the presence of low levels of lysine. Mild oxidative conditions were used to convert selenalysine into dehydroalanine post-translationally. Using this method, we demonstrate the preparation of polyunsaturated and highly decorated peptides. This report is an important step toward the preparation and selection of large libraries of protein-reactive compounds with potential use as novel drugs or as analytical tools.

Combined cold, acid, ethanol shocks in Oenococcus oeni: Effects on membrane fluidity and cell viability

The effects of combined cold, acid and ethanol on the membrane physical state and on the survival of Oenococcus oeni were investigated. Membrane fluidity was monitored on intact whole O. oeni cells subjected to single and combined cold, acid and ethanol shocks by using fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Results showed that cold shocks (14 and 8 °C) strongly rigidified plasma membrane but did not affect cell survival. In contrast, ethanol shocks (10–14% v/v) induced instantaneous membrane fluidisation followed by rigidification and resulted in low viability. Acid shocks (pH 4.0 and pH 3.0) exerted a rigidifying effect on membrane without affecting cell viability. Whatever the shock orders, combined cold (14 °C) and ethanol (14% v/v) shocks resulted in strong membrane rigidification. Interestingly, O. oeni survived combined cold and ethanol shocks more efficiently than single ethanol shock. Membrane rigidification was induced by ethanol-and-acid (10% v/v - pH 3.5) shock and correlated with total cell death. In contrast, O. oeni recovered its viability when subjected to cold (8 °C)-then-ethanol-and-acid shock which strongly rigidified the membrane. Our results suggested a positive short-term effect of combined cold, acid and ethanol shocks on membrane fluidity and viability of O. oeni.

Pyridone Dipeptides – Synthesis of a Novel Peptide Chromophore

AbstractSequential elimination of two water molecules transfers the diols 2 and 3 into the pyridone dipeptide 1. Both dehydration steps become part of the peptide synthesis under appropriate reaction conditions. A straightforward strategy for the incorporation of 1 in oligopeptide sequences is presented here. As part of the oligopeptide backbone, 1 has a local rigidifying effect which was studied in the cyclic octapeptides 16 and 25. Both cyclopeptides exhibit conformational averaging although a fixed conformation was identified for the 1‐Phe tripeptide fragment of octapeptide 25. The pyridone dipeptide 1 serves also as a fluorescence label and the UV/Vis‐spectroscopic properties of tetrapeptide 13 are investigated here. The peptide backbones of the two cyclic octapeptides 16 and 25 arrange a pair of chromophores 1 in close vicinity of each other. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Effect of a laminin amphiphatic sequence on DPPC ordered bilayers

A peptide sequence, stearoyl-GESIKVAVS(NH2), related to a laminin fragment, has been synthesized. Formation of aggregates was controlled by titrating a sodium anilinonaphthalene sulphonate (ANS) solution with peptide and recording fluorescence intensity increases. The results show that this system experiences a sudden increase in fluorescence at peptide concentrations around 2.5 x 10(-4) mol/L. The interaction of this hydrophobic peptide with DPPC vesicles has been studied using fluorescence techniques. Its influence on the microviscosity of bilayers was determined by studying polarization/temperature dependence for ANS and diphenyl hexatriene (DPH) fluorescent probes. With both markers the presence of peptide promotes a clear increase in anisotropy values. This indicates a rigidifying effect. Leakage studies carried out with liposomes loaded with carboxyfluorescein (CF) indicate a stabilizing effect of the peptide on bilayers, in agreement with results obtained with fluorescent probes.

Cholesterol reduces membrane electroporation and electric deformation of small bilayer vesicles

Electric fields, similar in the order of magnitude of the natural membrane fields of cellular lipid/protein membranes, and chemical relaxation spectrometry can be used as tools to quantify the rigidifying effect of cholesterol in membranes. Small unilamellar vesicles of radius a = 50 ± 3 nm, prepared form phosphatidylcholine, phosphatidylserine and phosphatidyl-glycerol in the molar ratio 1:1:1 and containing the optical lipid probe molecule 2-(3-diphenyl-hexatrienyl) propanoyl)-1-palmitoyl- sn-glycerol-3-phosphocholine (β-DPH pPC), serve as examples for curved lipid membranes. The data of electrooptical turbidity and absorbance relaxations at the wavelength λ = 365 nm are analysed in terms of membrane bending rigidity κ and membrane stretching modulus K. Both κ and K increase with increasing mole fraction x of cholesterol up to x = 0.5. The cholesterol induced denser packing of the lipids reduces the extent of both membrane electroporation (ME) and electroelongation of the vesicles. Further on, cholesterol in the lipid phase and sucrose in the aqueous suspension reduce the extent of membrane undulation and electro-stretching.

Probing nucleotide-binding effects on backbone dynamics and folding of the nucleotide-binding domain of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase.

In muscle cells, SERCA (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase) plays a key role in restoring cytoplasmic Ca2+ levels to resting concentrations after transient surges caused by excitation-coupling cycles. The mechanism by which Ca2+ is translocated to the lumen of the ER (endoplasmic reticulum) involves major conformational rearrangements among the three cytoplasmic domains: actuator (A), nucleotide-binding (N) and phosphorylation (P) domains; and within the transmembrane Ca2+-binding domain of SERCA. CD, fluorescence spectroscopy and NMR spectroscopy were used in the present study to probe the conformation and stability of the isolated N domain of SERCA (SERCA-N), in the presence and absence of AMP-PNP (adenosine 5'-[beta,gamma-imido]triphosphate). CD and tryptophan fluorescence spectroscopy results established that the effects of nucleotide binding were not readily manifested on the global fold and structural stability of SERCA-N. 15N-backbone-relaxation experiments revealed site-specific changes in backbone dynamics that converge on the central beta-sheet domain. Nucleotide binding produced diverse effects on dynamics, with enhanced mobility observed for Ile369, Cys420, Arg467, Asp568, Phe593 and Gly598, whereas rigidifying effects were found for Ser383, Leu419, Thr484 and Thr532. These results demonstrate that the overall fold and backbone motional properties of SERCA-N remained essentially the same in the presence of AMP-PNP, yet revealing evidence for internal counter-balancing effects on backbone dynamics upon binding the nucleotide, which propagate through the central beta-sheet.

Cholesterol modulates interaction between an amphipathic class A peptide, Ac-18A-NH2, and phosphatidylcholine bilayers.

Cholesterol (Chol) in phosphatidylcholine large unilamellar vesicles (PC LUV) modulated interaction of the bilayers with a class A amphipathic peptide, Ac-18A-NH2: Chol increased the peptide binding capacity and reduced the affinity together with the peptide-induced leakage of calcein from LUV. Similar effects of Chol have been observed on the interaction of LUV with apoA-I [Saito, H., Miyako, Y., Handa, T., and Miyajima, K. (1997) J. Lipid Res. 38, 287-294]. Circular dichroism (CD) spectra of the peptide indicated a similar helical structure formation in LUV with and without Chol. The fluorescence spectral shift, quantum yield, anisotropy, and acrylamide-quenching of the peptide Trp indicated that in PC:Chol (3:2) LUV, Ac-18A-NH2 was located in a more polar membrane environment with increased motional freedom and greater accessibility to the aqueous medium. Fluorescence energy transfer from the Trp indole ring to acceptors situated at different depths in the bilayers revealed that the amphipathic peptide penetrated the hydrophobic interior of PC bilayers, while the peptide was located at the polar zwitterionic surface in PC:Chol LUV. The inclusion of Chol causes the headgroup separation of PC at the surface of LUV and increases the binding maximum of the wedge-shaped amphipathic peptide without disrupting the membrane structure. In addition, the rigidifying effect of Chol on PC acyl chains prevents the penetration of the peptide into the bilayer interior. These findings imply that Chol in membranes affects the binding and motional freedom of exchangeable plasma apolipoproteins containing class A amphipathic sequences, e.g., apoA-I and apoCs.

Flexibility and packing in proteins.

Structural flexibility is an essential attribute, without which few proteins could carry out their biological functions. Much information about protein flexibility has come from x-ray crystallography, in the form of atomic mean-square displacements (AMSDs) or B factors. Profiles showing the AMSD variation along the polypeptide chain are usually interpreted in dynamical terms but are ultimately governed by the local features of a highly complex energy landscape. Here, we bypass this complexity by showing that the AMSD profile is essentially determined by spatial variations in local packing density. On the basis of elementary statistical mechanics and generic features of atomic distributions in proteins, we predict a direct inverse proportionality between the AMSD and the contact density, i.e., the number of noncovalent neighbor atoms within a local region of approximately 1.5 nm(3) volume. Testing this local density model against a set of high-quality crystal structures of 38 nonhomologous proteins, we find that it accurately and consistently reproduces the prominent peaks in the AMSD profile and even captures minor features, such as the periodic AMSD variation within alpha helices. The predicted rigidifying effect of crystal contacts also agrees with experimental data. With regard to accuracy and computational efficiency, the model is clearly superior to its predecessors. The quantitative link between flexibility and packing density found here implies that AMSDs provide little independent information beyond that contained in the mean atomic coordinates.

Astaxanthin and Peridinin Inhibit Oxidative Damage in Fe2+-Loaded Liposomes: Scavenging Oxyradicals or Changing Membrane Permeability?

Astaxanthin and peridinin, two typical carotenoids of marine microalgae, and lycopene were incorporated in phosphatidylcholine multilamellar liposomes and tested as inhibitors of lipid oxidation. Contrarily to peridinin results, astaxanthin strongly reduced lipid damage when the lipoperoxidation promoters—H2O2, tert-butyl hydroperoxide (t-ButOOH) or ascorbate—and Fe2+:EDTA were added simultaneously to the liposomes. In order to check if the antioxidant activity of carotenoids was also related to their effect on membrane permeability, the peroxidation processes were initiated by adding the promoters to Fe2+-loaded liposomes (encapsulated in the inner aqueous solution). Despite that the rigidifying effect of carotenoids in membranes was not directly measured here, peridinin probably has decreased membrane permeability to initiators (t-ButOOH > ascorbate > H2O2) since its incorporation limited oxidative damage on iron-liposomes. On the other hand, the antioxidant activity of astaxanthin in iron-containing vesicles might be derived from its known rigidifying effect and the inherent scavenging ability.

The role of retinal in the long-range protein-lipid interactions in bacteriorhodopsin-phosphatidylcholine vesicles

The effects of bacteriorhodopsin analogues and the analogues of a bacteriorhodopsin mutant (D96N) on the lateral organization of lipids have been investigated with lipid species with a variety of acyl chain lengths. The analogues, obtained by regeneration of bacterioopsin or mutant opsin with 14-, 12-, 10-, or 8-fluororetinal, were reconstituted with 1,2-didodecanoyl-sn-glycero-3-phosphocholine, 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine, 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, and 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine. The phase behavior of the protein-lipid systems was investigated at different temperatures and different protein/lipid molar ratios by analyzing the fluorescence and phase properties of the 1-acyl-2-[8-(2-anthroyl)octanol]-sn-glycero-3-phosphocholine probe. The (8,10,12)-bacteriorhodopsins had a similar effect on the lipid phase transition to that induced by native bacteriorhodopsin: a rigidifying effect on the three shorter lipid species and a fluidifying effect on the longest-chain lipids used. The substitution of retinal with 14-fluororetinal resulted in much stronger effects of the protein on the lipids: a more pronounced up-shift of the lipid phase transition temperature, a rigidifying effect on all the lipids used, and an elongation of the distance over which the hydrophobic thickness of the lipid bilayer was perturbed by the protein. Evidence was provided that retinal contributed to the long-range protein-lipid interactions in bacteriorhodopsin-phosphatidylcholine vesicles. The extent of this contribution was dependent on the retinal structure in close vicinity to the Shiff base and on the compactness of the protein structure.

Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa.

Semen cryopreservation is an important procedure in the treatment of human infertility. However, the ability of spermatozoa to survive freeze/thaw processes varies between patients. Cryopreservation-induced stress may result in membrane injury with consequent loss of sperm motility and viability. We investigated the relationship between the physico-chemical state of the human sperm membranes and their tolerance to cryopreservation. Conventional characteristics of 20 semen samples were analysed before and after cryopreservation as well as their membrane fluidity assessed by measuring the fluorescence polarization anisotropy, which is inversely proportional to the fluidity. Correlation between fluidity and post-thaw recoveries of motile and viable spermatozoa were examined. Results showed that membrane anisotropy markedly varies between patients. In cryopreserved spermatozoa, anisotropy values were significantly higher than in fresh spermatozoa. Furthermore, recovery of motile and viable spermatozoa after freeze/thaw was strongly correlated with anisotropy of fresh spermatozoa (P < 0.05). The higher the membrane fluidity was before freezing, the better was the response of spermatozoa to cryopreservation. The results indicate that the freeze/thaw process results in a rigidifying effect on the sperm membrane and suggest that sperm adaptability to freeze/thaw-induced stress could be dependent on their initial membrane fluidity. The latter finding has practical implications for predicting the response of spermatozoa following freezing and thawing and for improving the recovery of viable spermatozoa.

Rigidified calixarenes bearing four carbamoylmethylphosphineoxide or carbamoylmethylphosphoryl functions at the wide rim

Conformationally rigidified tetraCMPO derivatives have been prepared from calix[4]arene bis(crown ether) 4a in which adjacent oxygens are bridged at the narrow rim by two diethylene glycol links. Acylation of the tetraamine 4c with the CMPO-active ester 5b gave the tetraphosphine oxide 6a, while the tetraphosphinate 6b and the tetraphosphonate 6c were obtained by Arbuzov reaction of tetrabromoacetamido derivative 7 with PhP(OEt)2 or P(OEt)3. The extraction ability of these CMPO derivatives was checked for selected lanthanides and actinides and compared with the analogous compounds 1b, 10b and 10d derived from calix[4]arene tetrapentyl ether. All rigidified bis(crown ether) ligands are more effective extractants than their pentyl ether counterparts and require only 1/10 of the concentration (cL= 10 4M) to obtain the same distribution coefficients, while with CMPO itself a 2,000-fold concentration is necessary. This could be a consequence of a better preorganisation of the ligating functions owing to the rigidity which on the other hand did not change the observed selectivity for americium (DAm/DEu=9-19) and for light lanthanides over heavy ones. NMR relaxivity titration curves show that the complex of Gd3+ with ligand 6a is highly oligomerised in anhydrous acetonitrile over a large range of ligand:metal concentration ratios. Nuclear magnetic relaxation dispersion (NMRD) profiles also showed that large oligomers were formed, and their mean tumbling times were deduced from the Solomon-Bloembergen-Morgan equations. The NMR spectra of dia- and paramagnetic lanthanide complexes with 6a agreed with the presence of two conformers with an elongated calix[4]arene skeleton in which the distances between opposite methylene groups are different. Contrary to what was observed with ligand 2a, the addition of nitrate ions does not labilize the metal complexes, presumably because of the rigidification effect of the ether bridges. Single-crystal X-ray structures were obtained for the active ester 5b and for diphenylphosphorylacetic acid 5a.

Fatty acids specifically related to the anisotropic properties of plasma membrane from rat urothelium

Four different luminal surfaces of rat urothelium differing in their fatty acid composition were prepared by dietary induction. In order to induce lipid changes, each of four groups of rat received a basal diet rich in one of the unsaturated n-3, n-6 or n-9 fatty acid families and a commercial (control) diet. The effects of the dietary regime on the fatty acid composition of luminal urothelial membranes and their relation to the mobility of fluorescent probes were studied. In comparison with the control diet membrane, all three fatty acid-rich diets induced a decrease of the percentage amount of saturated fatty acid while that of the unsaturated fatty acids was increased. Accordingly, all three diets increased the unsaturation index in comparison with the control diet. The anisotropy across each membrane fraction was assessed using the n-(9-anthroyloxy) fatty acid fluorescent probes 3-AS, 7-AS and 12-AS, which locate at different depths in the membrane. Two different anisotropy profiles were observed. One profile showed the highest anisotropy at the C7 depth, whereas the other exhibited a continuous decrease of the anisotropy from the surface to the center of the bilayer. The molecular properties (isomerization) of 18:2 n-9 fatty acid may account, at least in part, for the observed V-shaped profile (the ascending trend) of the membrane anisotropy values as a function of the respective 18:2 n-9 fatty acid contents. Nevertheless, the minimum value of the profile did not correspond to the minimum 18:2 n-9 fatty acid content, but rather to the higher amount of docosahexaenoic (22:6 n-3) fatty acid. Thus, a modulating role of the 22:6 n-3 fatty acid on the rigidifying effect of 18:2 n-9 fatty acid is suggested, possibly mediated by relationships between fatty acid composition, saturated and unsaturated chain lengths, and freedom of motion of the phospholipid acyl chains.

Physicochemical Behavior of Polylysine-[HAV-VP3 Peptide] Constructs at the Air−Water Interface

Branched-chain polypeptides based on a polylysine backbone were synthesized for use as delivery systems for biologically active molecules. To develop a new immunoadjuvant system for hepatitis A virus (HAV) antigens, a peptide fragment (110−121) of the VP3 hepatitis A protein has been linked to cationic and amphoteric polymeric polypeptides. In the present paper, we describe the physicochemical characterization of three branched polypeptide-[HAV-VP3(110−121) peptide] constructs, where the peptide is attached to the polymer side chains by disulfide linkage. The surface activity of these three conjugates has been studied as a function of time and concentration in the subphase. Moreover, its insertion into phospholipid DPPC (dipalmitoylphosphatidylcholine) monolayers has also been determined. The results show that these constructs are surface active and can insert into the lipid monolayers. AK construct is slightly more active than EAK and SAK constructs both in the presence and absence of DPPC monolayers. This behavior suggests that construct disposition at interfaces is mainly dependent on nonsubstituted side chains. Fluorescence polarization studies, performed with DPPC vesicles saturated with either DPH (1,6-diphenyl-1,3,5-hexatriene) or ANS (1-anilino-8-naphthalenesulfonic acid), indicate a strong rigidifying effect of the three constructs on the polar heads and alkyl chains of bilayers.

Molecular rearrangements of thylakoids after heavy metal poisoning, as seen by Fourier transform infrared (FTIR) and electron spin resonance (ESR) spectroscopy

The specific effects exerted by different heavy metals on both the function and the structure of the photosynthetic apparatus were addressed. The functional analysis performed via the fluorescence induction kinetics revealed that the applied toxic heavy metals can be classified into two groups: Cd and Ni had no significant effect on the photosynthetic electron transport, while Cu, Pb and Zn strongly inhibited the Photosystem II (PS II) activity, as evidenced by the dramatic decreases in both the variable (Fv) and the maximal (Fm) fluorescence. The structural effects of the heavy metal ions on the thylakoid membranes were considered in three relations: (1) lipids, (2) proteins — studied by Fourier transform infrared (FTIR) spectroscopy, and (3) lipid—protein interactions — investigated by electron spin resonance (ESR) spectroscopy using spin-labeled probe molecules. The studied heavy metal ions had only a non-specific rigidifying effect on the thylakoid lipids. As regards proteins, Cd and Ni had no effect on the course of their heat denaturation. The heat denaturation of the proteins was accompanied by a decrease in the α-helix content (1656 cm-1), a parallel increase in the disordered segments (1651 cm-1), a decrease in the intramolecular β-sheet (1636 cm-1) content and the concomitant appearance of an intermolecular β-structure (1621 cm-1). In contrast with Cd and Ni, Cu and Zn blocked the appearance of the intermolecular β-structure. Pb represented an intermediate case. It seems that these heavy metals alter the native membrane structure in such a way that heat-induced aggregation becomes more limited. The ESR data revealed that certain heavy metals also affect the lipid—protein interactions. While Cd and Ni had hardly any effect on the solvation fraction of thylakoid lipids, Cu, Pb and Zn increased the fraction of lipids solvating the proteins. On the basis of the FTIR and ESR data, it seems that Cu, Pb, and Zn increase the surfaces available for lipid—protein interactions by dissociating membrane protein complexes, and that these ‘lipidated’ proteins have a smaller chance to aggregate upon heat denaturation. The data presented here indicate that the damaging effects of poisonous heavy metals are element-specific, Cu, Pb and Zn interact directly with the thylakoid membranes of the photosynthetic apparatus, while Cd and Ni interfere rather with other metabolic processes of plants.

A fluorescence and CD study on the interaction of synthetic lipophilic hepatitis B virus preS(120-145) peptide analogues with phospholipid vesicles.

The interaction of the immunogenic peptide of human hepatitis B virus (HBV) preS(120-145), including B and T epitopes, with phospholipid vesicles has been studied by fluorescence techniques and CD. In addition, interaction of three lipopeptides derived from preS(120-145) containing stearoyl, cholanoyl, and tripalmitoyl-S-glyceryl-cysteine (Pam3C) SS moieties with dipalmitoylphosphatidylcholine (DPPC) has been investigated by polarization fluorescence spectroscopy. Fluorescence experiments showed an increase in fluorescence intensity and a blue shift of the maximum emission wavelength upon interaction of preS(120-145) with DPPC vesicles below the transition temperature (Tc), indicating that the tryptophan moiety enters a more hydrophobic environment. Moreover, fluorescence polarization experiments showed that the peptide decreased the membrane fluidity at the hydrophobic core, increasing the Tc of the lipid and decreasing the amplitude of the change of fluorescence polarization associated with the cooperative melting of 1,6-diphenyl-1,3,5-hexatriene labeled vesicles. The absence of leakage of vesicle-entrapped carboxyfluorescein indicates that the peptide did not promote vesicle lysis. Besides, the three lipopeptides derived from preS(120-145) showed a more pronounced rigidifying effect at the hydrophobic core of the bilayer, with a significative increase in the Tc. Stearoyl- and cholanoyl-preS(120-145) restricted the motion of lipids also at the polar surface, whereas Pam3CSS-preS(120-145) did not alter the polar head group order. Finally, CD studies in 2,2,2-trifluoroethanol or in presence of vesicles suggested that the bound peptide adopted amphiphilic alpha-helical and beta-sheet structures, with an important contribution of the beta-turn. It is concluded that preS(120-145) can interact with the lipid membrane through the formation of an amphipathic structure combination of beta-sheet and alpha-helix aligned parallel to the membrane surface, involving the N-terminal residues, and penetrating only a short distance into the hydrophobic core. The C-terminal part, with a combination of beta-turn and beta-sheet structure, remains at the outer part of the bilayer, being potentially accessible to immunocompetent cells. Furthermore, coupling of an hydrophobic moiety to the N-terminal part of the peptide favors anchoring to the membrane, probably facilitating interaction of the peptide with the immunoglobulin receptor. These results are in agreement with the induction of immune response by preS(120-145) and with the enhanced immunogenicity found in general for lipid-conjugated immunopeptides.