Detergent Molecules Research Articles

New process for the production of branched‐chain fatty acids

AbstractFatty acids are the primary natural starting materials for the production of surfactant and detergent molecules. Derived from vegetable (oil) and animal (fat) triglyceride sources, the fatty acids are linear aliphatic carboxylic acids. Industrial interest in branched‐chain fatty acids is driven by the needs for products with enhanced performance benefits including higher solubility, ease of handling, better hard water tolerance, and improved oxidative stability. Therefore, catalytic processes have been developed for the conversion of linear fatty acids to branched ones. High yields of branched acids are obtained from unsaturated fatty acids over acidic zeolites, particularly those with large pores. Fatty esters also are readily isomerized to branched ones by means of the same catalyst and process. It is postulated that the isomerization of fatty acids proceeds through three‐ and four‐membered ring carbocation intermediates formed inside the largepore zeolites. This is supported by evidence of methyl and ethyl branching in the primary and final products. The total number of carbon atoms in the fatty acid molecule is unchanged. Surfactants derived from branched fatty acids show favorable physical properties, including a lower viscosity and improved handling, even as intended performance characteristics are maintained.

Thermodynamic and Kinetic Study of the Sphere-to-Rod Transition in Nonionic Micelles: Aggregation and Stress Relaxation in C14E8 and C16E8/H2O Systems

The energetics and dynamics of the growth of nonionic n-alkyl poly(ethylene glycol) ether surfactant micelles in the isotropic L 1 phase have been analyzed on the basis of a recently described, concentration dependent thermal transition. 1 This highly cooperative endothermic transition is assigned as a sphere-to-rod transition. We analyze the thermodynamic data in terms of aggregation-fusion of complete micelles and also discuss the kinetics in the frame of this random micelle model. The analysis of the calorimetric data leads to a cooperative unit of about 160 detergent molecules undergoing simultaneously the transition from spheroidal to cylindrical structure. We investigate the aggregation kinetics in the microsecond to millisecond range using the temperature-jump (TJ) method with observation of scattered and transmitted light and the stress relaxation dynamics monitoring transient electric birefringence (TEB). As expected from the model, the aggregation dynamics gets faster with increasing temperature, while the stress relaxation dynamics slows down with temperature, leading to apparent negative activation energies. At the transition temperatures, the dynamics of the structural transition for C 1 4 E 8 is characterized by an association and dissociation rate constant of 4.4 x 10 6 M - 1 s - 1 and 1.8 x 10 3 s - 1 , respectively, compared to the much slower dynamics of C 1 6 E 8 with the corresponding rate constants 8.5 x 10 4 M - 1 s - 1 and 29 s - 1 , respectively. The rate constants of micelle fusion increase, while those of micelle scission decrease with increasing temperature. The TEB results are discussed using the relations derived in the literature for the competition between rotational motion and chain scission. The fast dynamics of C 1 4 E 8 compared to the much slower dynamics of C 1 6 E 8 provides an understanding of the differences in stress compliance and viscosity for these systems. As a major result of the study, we present a consistent mechanism of micellar growth which involves the aggregation-fusion of two micelles in a single, rate-limiting reaction step and not the stepwise incorporation of single surfactant molecules into pre-existing micelles. The speeding up of the TJ kinetics in C 1 6 E 8 at higher temperatures indicates a further structural transition, possibly network formation.

Comparative Analyses of Three-Dimensional Models of Bacterial Reaction Centers

Despite the fact that the three-dimensional structure of an integral membrane protein was first determined 20 years ago, structures have been solved for very few membrane proteins. The reaction center is an exception with many mutant and modified structures available from 3 different bacterial species. In order to relate these structures to the function of the reaction center, an accurate assessment of the reliability of the structural models is required. Here we describe the quality of the structures of the bacterial reaction center based upon different criteria, such as evaluation of the geometry of the models and comparison of different models. Overall, the structures are found to be most accurate in the membrane-embedded regions with the periplasmic and cytoplasmic exposed regions having more disorder and differences among the structural models. In general, the cofactors and the surrounding protein regions are among the most accurately determined regions of the protein, except for the secondary quinone and its binding pocket that shows a large variation among structures. The limited accuracy of the secondary quinone is due to its partial occupancy as a consequence of its functional role and to the presence of surface features, including lipids and detergent molecules.

The 2.0 Å Resolution Crystal Structure of Prostaglandin H 2 Synthase-1: Structural Insights into an Unusual Peroxidase

Prostaglandin H 2 synthase (EC 1.14.99.1) is an integral membrane enzyme containing a cyclooxygenase site, which is the target for the non-steroidal anti-inflammatory drugs, and a spatially distinct peroxidase site. Previous crystallographic studies of this clinically important drug target have been hindered by low resolution. We present here the 2.0 Å resolution X-ray crystal structure of ovine prostaglandin H 2 synthase-1 in complex with α-methyl-4-biphenylacetic acid, a defluorinated analog of the non-steroidal anti-inflammatory drug flurbiprofen. Detergent molecules are seen to bind to the protein's membrane-binding domain, and their positions suggest the depth to which this domain is likely to penetrate into the lipid bilayer. The relation of the enzyme's proximal heme ligand His388 to the heme iron is atypical for a peroxidase; the iron–histidine bond is unusually long and a substantial tilt angle is observed between the heme and imidazole planes. A molecule of glycerol, used as a cryoprotectant during diffraction experiments, is seen to bind in the peroxidase site, offering the first view of any ligand in this active site. Insights gained from glycerol binding may prove useful in the design of a peroxidase-specific ligand.

Dissipative structures formed in the course of drying the aqueous solution of n -dodecyltrimethylammonium chloride on a cover glass

Macroscopic and microscopic dissipative structural patterns are formed in the course of drying an aqueous solution of n-dodecyltrimethylammonium chloride on a cover glass. Broad ring patterns of the hill accumulate with detergent molecules to form around the outside edges of the film solution in the macroscopic scale. The drying time (T) and the pattern area (S) decrease and increase respectively, as the detergent concentration increases. T decreases significantly as the ethanol fraction increases in the aqueous ethanol mixtures, whereas S increases as the fraction increases. Both T and S decrease as the concentrations of KCl, CaCl2 or LaCl3 increase. Cross-, branch-, and arc-like microscopic patterns are observed in the separated block regions. The convection of water and detergents at different rates under gravity and the translational and rotational Brownian movement of the latter are important for macroscopic pattern formation. Microscopic patterns are determined by the translational Brownian diffusion of the detergent molecules and the electrostatic and the hydrophobic interactions between the detergents and/or between the detergent and cell wall in the course of the solidification.

Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus.

Catalysis of glutamate transport across cell membranes and coupling of the concentrative transport to sodium, proton, and potassium gradients are processes fundamental to organisms in all kingdoms of life. In bacteria, glutamate transporters participate in nutrient uptake, while in eukaryotic organisms, the transporters clear glutamate from the synaptic cleft. Even though glutamate transporters are crucial to the viability of many life forms, little is known about their structure and quaternary organization. In particular, the subunit stoichiometry of these polytopic integral membrane proteins has not been unequivocally defined. Determination of the native molecular mass of membrane proteins is complicated by their lability in detergent micelles and by their association with detergent and/or lipid molecules. Here we report the purification of glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus in a monodisperse, detergent-solubilized state. Characterization of both transporters either by chemical cross-linking and mass spectrometry or by size-exclusion chromatography and in-line laser light scattering, refractive index, and ultraviolet absorption measurements shows that the transporters have a trimeric quaternary structure. Limited proteolysis further defines regions of primary structure that are exposed to aqueous solution. Together, our results define the subunit stoichiometry of high-affinity glutamate transporters from B. caldotenax and B. stearothermophilus and localize exposed and accessible elements of primary structure. Because of the close amino acid sequence relationship between bacterial and eukaryotic transporters, our results are germane to prokaryotic and eukaryotic glutamate and neutral amino acid transporters.

Crystal structure of UDP-N-acetylglucosamine acyltransferase from Helicobacter pylori.

Crystal structure of UDP-N-acetylglucosamine acyltransferase from Helicobacter pylori.

Importance of the lid and cap domains for the catalytic activity of gastric lipases

Human gastric lipase (HGL) is an enzyme secreted by the stomach, which is stable and active despite the highly acidic environment. It has been clearly established that this enzyme is responsible for 30% of the fat digestion processes occurring in human. This globular protein belongs to the α/ β hydrolase fold family and its catalytic serine is deeply buried under a domain called the extrusion domain, which is composed of a ‘cap’ domain and a segment consisting of 58 residues, which can be defined as a lid. The exact roles played by the cap and the lid domains during the catalytic step have not yet been elucidated. We have recently solved the crystal structure of the open form of the dog gastric lipase in complex with a covalent inhibitor. The detergent molecule and the inhibitor were mimicking a triglyceride substrate that would interact with residues belonging to both the cap and the lid domains. In this study, we have investigated the role of the cap and the lid domains, using site-directed mutagenesis procedures. We have produced truncated mutants lacking the lid and the cap. After expressing these mutants and purifying them, their activity was found to have decreased drastically in comparison with the wild type HGL. The lid and the cap domains play an important role in the catalytic reaction mechanism. Based on these results and the structural data (open form of DGL), we have pointed out the cap and the lid residues involved in the binding with the lipidic substrate.

Direct analysis of protein sedimentation equilibrium in detergent solutions without density matching

Characterizing membrane proteins by sedimentation equilibrium is challenging because detergents and/or lipid molecules, usually required for solubilization, form a complex with the protein. The most common way to overcome this problem is Tanford and Reynolds’ density matching method, which eliminates the buoyant mass contributions of detergents/lipids by adjusting the solvent density with D 2O/H 2O mixtures to render either detergent or lipid molecules neutrally buoyant. Unfortunately, the method is practical only for detergent densities between 1.0 (H 2O) and 1.1 (D 2O) g ml −1, excluding many of the more commonly used detergents for membrane protein studies. Here, we present a modern variant of Tanford and Reynolds’ method that (1) is applicable to any detergent regardless of its specific density, (2) does not compromise accuracy and precision, and (3) provides additional information about the number of detergent molecules that are bound to each protein. The new method was applied successfully to Δ(1-43)A-I, an amino-terminal deletion mutant of human apolipoprotein A-I. Interestingly, we observed a significantly lower Δ(1-43)A-I/octyl-glucoside complex partial specific volume than that expected from volume additivity rules, indicative of specific protein–detergent interactions.

Crystal Structure of Neisserial Surface Protein A (NspA), a Conserved Outer Membrane Protein with Vaccine Potential

The neisserial surface protein A (NspA) from Neisseria meningitidis is a promising vaccine candidate because it is highly conserved among meningococcal strains and induces bactericidal antibodies. NspA is a homolog of the Opa proteins, which mediate adhesion to host cells. Here, we present the crystal structure of NspA, determined to 2.55-A resolution. NspA forms an eight-stranded antiparallel beta-barrel. The four loops at the extracellular side of the NspA molecule form a long cleft, which contains mainly hydrophobic residues and harbors a detergent molecule, suggesting that the protein might function in the binding of hydrophobic ligands, such as lipids. In addition, the structure provides a starting point for structure-based vaccine design.

Line thermodynamics: adsorption at a membrane edge.

We report a novel experimental study of line thermodynamics. Our system consists of detergent molecules adsorbing at the edges of freestanding lipid bilayers. Adsorption reduces the line tension T of the membrane edges. Measuring T as a function of the bulk detergent concentration C, we obtain a line adsorption isotherm. Using an extension of Gibbs's surface thermodynamics to lines, we estimate the "line excess density" of adsorbants and the energy of adsorption per site.

The dual role of CHAPS in the crystallization of stromelysin-3 catalytic domain.

CHAPS [3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate] is a non-denaturing detergent widely used for protein solubilization and stabilization. CHAPS was used to avoid protein aggregation during concentration of the recombinant stromelysin-3 (ST3) catalytic domain and was required to stabilize the protein, allowing its crystallization. The crystal structure of the complex between the ST3 catalytic domain and a phosphinic inhibitor shows two CHAPS molecules binding to ST3 in two different orientations. One CHAPS molecule is masking a hydrophobic surface of the protein, thus avoiding protein aggregation. This detergent molecule is also involved in packing interactions. The other detergent molecule is located in a pocket formed by the N- and C-terminal parts of the ST3 and stabilizes a loop that normally binds a Ca atom.

Fluorescence Probe Study of Bicelle Structure as a Function of Temperature: Developing a Practical Bicelle Structure Model

Bilayered mixed micelles or bicelles are magnetically anisotropic, self-assembling model membrane structures comprised of long-chain phospholipids and short-chain detergent molecules. In the most widely accepted model of this system, the bicelle is discoid in shape, with the short-chain DHPC molecules aggregating to form rims around long-chain DMPC bilayers. While this model is consistent with most NMR and scattering data (X-ray and neutron), it inadequately describes the liquid-crystalline behavior of bicelle solutions at temperatures where magnetic alignment occurs. Temperature plays a central role in the structure of lipid aggregates, and the impact of temperature on bicelles has not been studied as extensively as composition and concentration. Therefore, a series of fluorescence probe and resonance energy transfer (FRET) measurements of labeled bicelle solutions as a function of temperature were conducted to monitor lipid mixing as an indication of bicelle structure and aggregation. The results of the...

Simulation and Modeling of the Rhodobacter sphaeroides Bacterial Reaction Center: Structure and Interactions

In this paper, we present the structural and dynamic results of a 3.4 ns molecular dynamics simulation of a reaction center protein immersed in a micelle-like environment formed by a detergent, lauryl dimethyl amino oxide or LDAO, and hydrated by more than 6000 additional water molecules. The whole system, ≈40 000 atoms, was simulated using an all-atom force field with refined potential parameters developed by us to describe the protein cofactors and the detergent molecules. LDAO, inserted at the beginning of the run in a configuration far from equilibrium, rearranged forming a micelle attached to the hydrophobic regions of the protein. The micelle is a stable and dynamic structure over the whole trajectory and prevents the protein and the internal cofactors from contacts with water. Comparing our simulated system with the high-resolution crystallographic structure, the deviations of the backbone atoms are small, less than 1.8 Å after 3.4 ns, and the chromophore arrangement geometry is stable and close to X-ray for the whole simulation. Thus, our system constitutes a realistic model to investigate at an atomic level the role of the protein environment on the charge transfer processes taking place in this complex. Related to the functionality of this RC protein, we observe the isomerization of the tyr M210 which is directly coupled with the primary electron transfer. Concerning the branch functionality of the RC, we observe that the computed minimum distance between the chromophores on the L and M side has a different dynamic behavior and is smaller on average for transitions on the L side. This finding might have some bearing on the electron-transfer asymmetry of the primary charge transfer.

Small-Angle Neutron Scattering Study of Micelle Structure in Protein-Detergent Complexes

Small-angle neutron scattering measurements have been carried out on micellar solutions of the detergent sodium dodecyl sulfate (SDS) and its complexes with the protein Bovine Serum Albumin (BSA) in aqueous solutions. It is found that the structure of micelle-like clusters bound to the protein in protein-detergent complexes is different from that of normal micelles in the pure detergent solutions. In protein-detergent complexes, the micelle-like clusters tend to be spherical with a much smaller aggregation number (detergent molecules per micelle) than those in normal micelles, and they are independent of the shape and aggregation number of the micelles in the corresponding pure detergent solutions.

The influence of detergents on the solubility of membrane proteins.

The relationship between the effect of detergents and amphiphiles on protein solubility and their use in crystallization solutions was examined for the reaction center from Rhodobacter sphaeroides. Measurement by a centrifugation assay of the solubility of the reaction center as a function of ionic strength revealed dramatic differences in the intrinsic solubility at zero ionic strength in the presence of various detergents and amphiphiles. High protein-solubility values were found for beta-octyl glucoside and for lauryldimethylamine-N-oxide with heptanetriol. The solubility differences are interpreted in terms of fundamental properties such as the polarity of the detergent molecules. Conditions that resulted in high protein solubility correspond to conditions that have been shown to be successful for crystallization of the reaction center. These results suggest that crystallization is favored for detergents and amphiphiles that optimize the solubility of integral membrane proteins.

Oligomeric States of the Detergent-solubilized Human Serum Paraoxonase (PON1)

Human plasma paraoxonase (HuPON1) is a high density lipoprotein (HDL)-bound enzyme exhibiting antiatherogenic properties. The molecular basis for the binding specificity of HuPON1 to HDL has not been established. Isolation of HuPON1 from HDL requires the use of detergents. We have determined the activity, dispersity, and oligomeric states of HuPON1 in solutions containing mild detergents using nondenaturing electrophoresis, size exclusion chromatography, and cross-linking. HuPON1 was active whatever its oligomeric state. In nonmicellar solutions, HuPON1 was polydisperse. In contrast, HuPON1 exhibited apparent homogeneity in micellar solutions, except with CHAPS. The enzyme apparent hydrodynamic radius varied with the type of detergent and protein concentration. In C(12)E(8) micellar solutions, from sedimentation velocity, equilibrium analytical ultracentrifugation, and radioactive detergent binding, HuPON1 was described as monomers and dimers in equilibrium. A decrease of the detergent concentration shifted this equilibrium toward the formation of dimers. About 100 detergent molecules were associated per monomer and dimer. The assembly of amphiphilic molecules, phospholipids in vivo, in sufficiently large aggregates could be a prerequisite for anchoring of HuPON1 and then allowing stabilization of the enzyme activity. Changes of HDL size and shape could strongly affect the binding affinity and stability of HuPON1 and result in reduced antioxidative capacity of the lipoprotein.

Structure of human CD1b with bound ligands at 2.3 A, a maze for alkyl chains.

The human genome encodes five nonpolymorphic major histocompatibility complex class I-like glycoproteins, CD1a to CD1e, that present lipid antigens for specific recognition by T lymphocytes. Using single alkyl chain detergents, we developed a protocol to generate recombinant human CD1b-lipid complexes. We present here the crystal structures of CD1b in complex with either phosphatidylinositol or ganglioside GM2 at 2.3 A and 2.8 A resolutions, respectively. The antigen-binding groove houses four interlinked hydrophobic channels that are occupied by the alkyl chains of the glycolipid plus two detergent molecules. A distinct exit beneath the alpha 2 helix further contributes to the plasticity of the binding groove. These structures reveal the mechanism by which two alkyl chain lipids bind to CD1b, and how CD1b can adapt to ligands of different alkyl chain length. They also suggest how very long alkyl chains, such as those of mycolic acid, could be fully contained within the binding groove. These results extend the spectrum of potential CD1b ligands by revealing that, in addition to two alkyl chain lipids, mono-alkyl and triple-alkyl chain lipids can be accommodated in the binding groove.

UV-Induced Gelation on Nanometer Scale Using Liposome Reactor

A protocol for preparation of polymer hydrogel spherical particles on a nanometer scale (nanogels) was developed. The protocol includes encapsulation of hydrogel-forming components into liposomes, UV-induced polymerization within the liposomes, solubilization of the lipid bilayer by detergent, removal of the phospholipid, detergent molecules, and their micelles by dialysis, and drying nanogels by evaporation in a temperature gradient. Dynamic light scattering technique was employed to characterize the size distribution of poly(acrylamide), poly(N-isopropylacrylamide), and poly(N-isopropylacrylamide-co-1-vinylimidazole) hydrogel particles. Hydrophobic chains of N-octadecylacrylamide were immobilized onto the surface of the poly(N-isopropylacrylamide-co-1-vinylimidazole) hydrogel particles to use them as anchors for attaching the nanogels onto the lipid bilayers. The diameter of the nanogels prepared varied between 30 and 300 nm. The solvent, temperature, and pH sensitivities of the liposomes, nanogels, and...

Molecular organization and dynamics of micellar phase of polyelectrolyte–surfactant complexes: ESR spin probe study

Molecular dynamics and organization of the micellar phase of complexes of linear polyelectrolytes with ionogenic and non-ionogenic surfactants was studied by the ESR spin probe method. Complexes of polyacrylic acid (PAA) and sodium polystyrenesulfonate (PSS) with alkyltrimethylammonium bromides (ATAB), as well as complexes of poly- N, N′-dimethyldiallylammonium chloride (PDACL) with sodium dodecylsulfate (SDS) were studied. The micellar phase of such complexes is highly organized molecular system, molecular ordering of which near the polymeric chain is much higher than in the ‘center’ of the micelle, it depends on the polymer–detergent interaction, flexibility of polymeric chain and length of carbonic part of the detergent molecule. Complexes of polymethacrylic acid (PMAA) with non-ionic detergent (dodecyl-substituted polyethyleneglycol), show that the local mobility of surfactant in such complexes is significantly lower than in ‘free’ micelles and depends on the number of micellar particles participating in formation of complexes.