Oxidation of α-tocopherol in micelles and liposomes by the hydroxyl,perhydroxyl, and superoxide free radicals

Rates of oxidation of α-tocopherol by the hydroxyl- and superoxide free radicals were measured. The radicals were produced in known yields by radiolysis of aqueous solutions with gamma rays. Two main systems were used to dissolve the tocopherol; micelles, made up from charged and uncharged amphiphiles, and membranes made from dimyristyl phosphatidylcholine which could be charged by addition of stearyl amine or dicetyl phosphate. The HO. radicals were efficient oxidants of α-tocopherol in all systems, with up to 83% of radicals generated in micelle and 32% in membrane suspensions initiating the oxidation. The HO_{$\text{2}\text{?}$} radical was an even more effective oxidant, but when most of it was in the O form at neutral or alkaline pH, the oxidation rates became low. Tocopherol held in positively charged micelles or membranes was oxidized at a higher rate by the O than in uncharged or negative particles. Possible biological significance of these results is discussed.     

Thermostability of α-chymotrypsin encapsulated in reversed micelles

Summary The influence of water content, additives, pH and substrate concentration on the thermostability of -chymotrypsin entrapped in a reversed micellar system of the cationic surfactant TTAB/heptane/ chloroform, was studied. Increasing the water level inside the reversed micelles diminishes the enzyme stability. Enzyme stability enhancement was achieved with the addition of glycerol, by increasing the nucleophile concentration or by decreasing the buffer pH.     

A negative staining study of natural and synthetic L-α-lysophosphatidylcholine micelles,macromolecular aggregates and crystals

A comparative assessment has been made by transmission electron microscopy of negatively stained specimens, of the micellar, aggregated and crystalline states of palmitoyl, oleoyl and ex ovo L-α-lysophosphatidylcholine present in aqueous suspensions. Micelle formation from dry lysophosphatidylcholines is shown to be temperature dependent. The presence of the unsaturated fatty acid in oleoyl L-α-lysophosphatidylcholine and some degree of unsaturation in L-α-lysophosphatidylcholine (ex ovo) promotes micelle formation at low temperatures (4 and 22°C), whereas crystalline palmitoyl L-α-lysophosphatidylcholine is essentially insoluble at low temperatures and requires incubation at 60°C to produce a micellar suspension.It is suggested that the micellar conformation is not spherical, a cylindrical or discoid shape is more compatible with the images presented. Both palmitoyl and ex ovo L-α-lysophosphatidylcholine produce flexibel rod-like micellar aggregates ca 6 nm in diameter and larger (20–60 nm dia) stacked-disc aggregates, again with a temperature dependency. The thickness of the disc-like L-α-lysophosphatidylcholine of a phospholipid bilayer (ca 6–7 nm). This, together with the ability of palmitoyl L-α-lysophosphatidylcholine to crystallize as multi-lamellar hexagonal particles which remain stable in aqueous suspensions at 4°C, suggests that, as with other phospholipids, the L-α-lysophosphatidylcholines possess the property of forming lamellar structures, but that these become increasingly unstable at higher temperatures depending on the fatty acid unsaturation. Ammonium molybdate and sodium phosphotungstate have been found to be more satisfactory than uranyl acetate for negative staining of aqueous suspensions of L-α-lysophosphatidylcholines.     

Bounded water kinetic model of β-galactosidase in reverse micelles

In the present investigation, -galactosidase was solubilized into Aerosol OT (AOT)/isooctane reverse micelles. Kinetic data for the hydrolysis of o-nitrophenyl--D-galactopyranoside (ONPG) at different pH values and molar ratios of water to AOT (W_o) were collected. It was observed that the usual kinetic model used for -galactosidase catalysis in aqueous systems failed to represent the experimental data. A bounded water model, however, showed a better correlation between enzymatic activity and W_o. In contrast to the aqueous system, controlling the water concentration in the reverse micelles allows the rate constants for the reaction between water molecules and glycosyl-enzyme complexes to be evaluated.     

Variability in secondary structure of the antimicrobial peptide Cateslytin in powder,solution, DPC micelles and at the air–water interface

Cateslytin (bCGA ³⁴⁴RSMRLSFRARGYGFR³⁵⁸), a five positively charged 15 amino-acid residues arginine-rich antimicrobial peptide, was synthesized using a very efficient procedure leading to high yields and to a 99% purity as determined by HPLC and mass spectrometry. Circular dichroism, polarized attenuated total reflectance fourier transformed infrared, polarization modulation infrared reflection Absorption spectroscopies and proton two-dimensional NMR revealed the flexibility of such a peptide. Whereas being mostly disordered as a dry powder or in water solution, the peptide acquires a α-helical character in the “membrane mimicking” solvent trifuoroethanol. In zwitterionic micelles of dodecylphophatidylcholine the helical character is retained but to a lesser extent, the peptide returning mainly to its disordered state. A β-sheet contribution of almost 100% is detected at the air–water interface. Such conformational plasticity is discussed regarding the antimicrobial action of Cateslytin. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Monomeric α-synuclein binds Congo Red micelles in a disordered manner

The histological dye Congo Red (CR) previously has been shown to inhibit α-synuclein (aS) fibrillation, but the mode of this inhibition remained unclear. Because of favorable exchange kinetics, interaction between CR and aS lends itself to a detailed nuclear magnetic resonance study, and relaxation dispersion measurements yield the bound fraction and time scales for the interaction of aS with CR. We find that at pH 6, CR exists as a micelle, and at a CR:aS molar ratio of ~1, only a small fraction of aS (~2%) is bound to these micelles. Rapid exchange (k(ex) ~ 3000 s(-1)) between the free and CR-bound states broadens and strongly attenuates resonances of aS by two processes: a magnetic field-dependent contribution, caused by the chemical shift difference between the two states, and a nearly field-independent contribution caused by slower tumbling of aS bound to the CR micelle. The salt dependence of the interaction suggests a predominantly electrostatic mechanism for the 60 N-terminal residues, while the weaker interaction between residues 61-100 and CR is mostly hydrophobic. Chemical shift and transferred NOE data indicate that aS becomes slightly more helical but remains largely disordered when bound to CR. Results indicate that inhibition of fibril formation does not result from binding of CR to free aS and, therefore, must result from interaction of aS fibrils or protofibrils with CR micelles.     

Self-replicating micelles — A chemical version of a minimal autopoietic system

Reverse micelles hosting the internal production of the surfactant are proposed as experimentally feasible models of simple (or minimal) autopoietic systems. We describe the conditions under which these may be formed and their possible biological implications. The micellar systems considered here turn out also to exhibit a capacity for self-reproduction through fragmentation under plausible conditions, thus constituting also a minimal experimental model for prebiotic self-reproduction.     

DNA polymerase β reveals enhanced activity and processivity in reverse micelles

Water is essential for the stability and functions of proteins and DNA. Reverse micelles are simple model systems where the structure and dynamics of water are controlled. We have estimated the size of complex reverse micelles by light scattering technique and examined the local microenvironment using fluorescein as molecular probe. The micelle size and water polarity inside reverse micelles depend on water volume fraction. We have investigated the different hydration and confinement effects on activity, processivity, and stability of mammalian DNA polymerase β in reverse micelles. The enzyme displays high processivity on primed single-stranded M13mp19 DNA with maximal activity at 10% of water content. The processivity and activity of DNA polymerase strongly depend on the protein concentration. The enzyme reveals also the enhanced stability in the presence of template-primer and at high protein concentration. The data provide direct evidence for strong influence of microenvironment on DNA polymerase activity.     

Amylose–dye complexes in cationic micelles: an optical spectroscopy study

The formation of amylose complexes with rose bengal (RB), erythrosine B (ER), and phenolphthalein (PP) in the presence of the cationic detergent tetradecyltrimethylammonium bromide (TTABr) was studied using optical spectroscopy methods. Absorption spectroscopy, steady-state fluorescence spectroscopy and picosecond time-resolved fluorescence spectroscopy were used to derive association constants k_s of the dyes, critical micelle concentration (CMC) values and structural information on the complexes formed. It seems that PP fits very well into amylose sites, where it forms an efficient inclusion complex with k_s=44,500 M⁻¹. The molecular diameter of RB is too big to fit the amylose cavity. Only part of the xanthene unit may be adopted in the helical cavity of amylose, whereas most of the interaction occurs through electrostatic and/or dipole–dipole interactions with the amylose chain. The ER molecule is an intermediate case, because it may fit the amylose cavity or adsorb on the amylose surface to form a complex. The presence of a surfactant in the amylose–ligand system increases the association constant for all dyes. In the presence of amylose, a decrease of the detergent CMC value of about one order of magnitude is observed. It is probable that the increased number of micelles incorporate more dyes into the amylose vicinity, which finally changes the structure of the amylose chain. On a macro scale, it was noted that the samples with dyes and detergent have a lower tendency to precipitate and the gelation process is delayed compared to that in water.     

Optimization of pH and temperature for β-xylosidase recovery by reversed micelles

A mathematical model was found to represent the enzyme yield (Y) as a function of pH (X1) and temperature (X2): Y=36.89+10.83X1–12.17X2–6X1X2–17.24 X12–13.24X22. The optimum values for pH and temperature to attain the maximum b-xylosidase recovery (42%) were 3.3 and 24 °C, respectively.     

NMR characterization of the interactions between lyso-GM1 aqueous micelles and amyloid β

Gangliosides are targets for a variety of pathologically relevant proteins, including amyloid β (Aβ), an important component implicated in Alzheimer’s disease (AD). To provide a structural basis for this pathogenic interaction associated with AD, we conducted NMR analyses of the Aβ interactions with gangliosides using lyso-GM1 micelles as a model system. Our NMR data revealed that the sugar-lipid interface is primarily perturbed upon binding of Aβ to the micelles, underscoring the importance of the inner part of the ganglioside cluster for accommodating Aβ in comparison with the outer carbohydrate branches that provide microbial toxin- and virus-binding sites.     

Localization of κ-casein on thin sections of casein micelles by the gold method

Summary The ultrastructural location of -casein in bovine casein micelles was investigated by the protein A-gold method. Casein micelles, fixed in glutaraldehyde, were embedded at low temperature to enhance immunocytochemical marking of thin sections. -Casein was found distributed throughout the micelles of all sizes with a higher concentration in the smaller micelles. No peripheral location of -casein was observed, even in the larger micelles. These results do not agree with coat-core structures proposed for casein micelles. However they favor models where -casein is distributed uniformly throughout the micelles.     

Nuclear magnetic resonance investigation of the conformation of δ-haemolysin bound to dodecylphosphocholine micelles

δ-Haemolysin in mixed micelles with perdeuterated dodecylphosphocholine was investigated with two-dimensional proton nuclear magnetic resonance experiments at 500 MHz. A single set of resonance lines was observed for the micelle-bound polypeptide, indicating that δ-haemolysin adopts a single conformation in this environment. Nearly complete, sequence-specific assignments were obtained for the segment 5–23 of this 26 residue polypeptide chain. From the sequential connectivities and numerous medium-range nuclear Overhauser effects this central portion of the molecule was found to form an extended helix with pronounced amphipathic distribution of polar and nonpolar amino acid side-chains.     

Study of the Alzheimer's Aβ40 peptide in SDS micelles using molecular dynamics simulations

The interaction of the Alzheimer's amyloid beta peptide, Aβ40, with sodium dodecyl sulfate (SDS) micelles, together with the self-assembly of SDS molecules around the peptide from an initial random distribution were studied using atomistic and coarse-grained (CG) molecular dynamics simulations. In atomistic simulations, the peptide structure in the micelle was characterized by two helical regions connected through a short hinge. The initial structure of the system was shown to affect the simulation results. The atomistic self-assembly of SDS molecules resulted in a 38-molecule micelle around the peptide, along with some globules and individual molecules. Coarse-grained simulation results, however, did not show such a difference, and at the end of all CG simulations, a complete 60-molecule micelle was obtained, with the peptide located at the interface of the micelle with water. The obtained CG radial density profiles and SDS micelle size and shape properties were identical for all CG simulations.     

Localization of glycosylated κ-casein on thin sections of casein micelles by lectin-labelled gold markers

Summary The location of the glycosylated part of κ-casein in bovine casein micelles was investigated using gold particles (6 nm in diameter) labelled withRicinus communis lectin andLimulus polyphemus lectin. The pattern of marking of thin sections of micelles was similar with both lectins. Glycosylated κ-casein was distributed uniformly throughout most micelles of all sizes. Peripheral location of glycosylated κ-casein was observed only occasionally in some of the largest micelles. Quantitative data indicated that the concentration of the glycosylated protein was constant in micelles of increasing sizes. As larger micelles contain less total κ-casein than smaller ones, these data indicated that a greater proportion of their κ-casein is glycosylated. These results support models for casein micelle structure where κ-casein is distributed throughout the micelles. They do not agree with “coat-core” structures.     

Lipid–protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: Comparison with detergent micelles,bicelles and liposomes

Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid–protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K⁺ channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~ 80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of ¹⁵N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.     

Structural characterization of Hsp12, the heat shock protein from Saccharomyces cerevisiae, in aqueous solution where it is intrinsically disordered and in detergent micelles where it is locally α-helical

Hsp12 (heat shock protein 12) belongs to the small heat shock protein family, partially characterized as a stress response, stationary phase entry, late embryonic abundant-like protein located at the plasma membrane to protect membrane from desiccation. Here, we report the structural characterization of Hsp12 by NMR and biophysical techniques. The protein was labeled uniformly with nitrogen-15 and carbon-13 so that its conformation could be determined in detail both in aqueous solution and in two membrane-mimetic environments, SDS and dodecylphosphocholine (DPC) micelles. Secondary structural elements determined from assigned chemical shifts indicated that Hsp12 is dynamically disordered in aqueous solution, whereas it gains four helical stretches in the presence of SDS micelles and a single helix in presence of DPC. These conclusions were reinforced by circular dichroism spectra of the protein in all three environments. The lack of long range interactions in NOESY spectra indicated that the helices present in SDS micelles do not pack together. R(1) and R(2), relaxation and heteronuclear NOE measurements showed that the protein is disordered in aqueous solution but becomes more ordered in presence of detergent micelles. NMR spectra collected in presence of paramagnetic spin relaxation agents (5DSA, 16DSA, and Gd(DTPA-BMA)) indicated that the amphipathic α-helices of Hsp12 in SDS micelles lie on the membrane surface. These observations are in agreement with studies suggesting that Hsp12 functions to protect the membrane from desiccation.     

Molecular dynamics simulations of helical antimicrobial peptides in SDS micelles: what do point mutations achieve?

We report long time scale simulations of the 18-residue helical antimicrobial peptide ovispirin-1 and its analogs novispirin-G10 and novispirin-T7 in SDS micelles. The SDS micelle serves as an economical and effective model for a cellular membrane. Ovispirin, which is initially placed along a micelle diameter, diffuses out to the water-SDS interface and stabilizes to an interface-bound steady state in 16.35 ns of simulation. The final conformation, orientation, and the structure of ovispirin are in good agreement with the experimentally observed properties of the peptide in presence of lipid bilayers. The simulation succeeds in capturing subtle differences of the membrane-bound peptide structure as predicted by solid state NMR. The novispirins also undergo identical diffusion patterns and similar final conformations. Although the final interface-bound states are similar, the simulations illuminate the structural and binding properties of the mutant peptides which make them less toxic compared to ovispirin. Based on previous data and the current simulations, we propose that introduction of a bend/hinge at the center of helical antimicrobial peptides (containing a specific C-terminal motif), without disrupting the helicity of the peptides might attenuate host-cell toxicity as well as improve membrane binding properties to bacterial cellular envelopes.     

Two-dimensional NMR study of the conformation of (34–65)bacterioopsin polypeptide in SDS micelles

Summary The conformation of the synthetic 32-residue polypeptide, an analog of the membrane spanning segment B (residues 34-65) ofHalobacterium halobium bacteriobpsin, incorporated into perdeuterated sodium dodecyl sulfate micelles in the presence of trifluoroethanol was investigated by¹H NMR spectroscopy. The spectrum resonances were assigned by means of phase-sensitive DQF-COSY, TOCSY and NOESY techniques. Interproton nuclear Overhauser effects and deuterium exchange rates of individual NH groups were derived from two-dimensional NMR spectra. Analysis of the obtained data showed that segment B has a right-handed a-helical stretch from Lys⁴¹ to Leu⁶² with a kink at Pros⁵⁰. The-helix in the C-terminal part is terminated at Gly⁶³, which adopts a conformation typical of amino acid residues in a left-handed helix. The N-terminal part (residues 34–40) has no ordered conformation. NMR data are provided for comparison of the segment B conformation in the isotropic system of an organic solvent, in SDS micelles and in the purple membrane bacterioopsin. Factors affecting the conformation of membrane spanning segment B in various milieus are discussed.Dedicated to the memory of Professor V.F. Bystrov