Cold denaturation of encapsulated ubiquitin

Theoretical considerations suggest that protein cold denaturation can potentially provide a means to explore the cooperative substructure of proteins. Protein cold denaturation is generally predicted to occur well below the freezing point of water. Here NMR spectroscopy of ubiquitin encapsulated in reverse micelles dissolved in low viscosity alkanes is used to follow cold-induced unfolding to temperatures below -25 degrees C. Comparison of cold-induced structural transitions in a variety of reverse micelle-buffer systems indicate that protein-surfactant interactions are negligible and allow the direct observation of the multistate cold-induced unfolding of the protein.     

Dynamics of low temperature induced water shedding from AOT reverse micelles

The effects of low temperature and ionic strength on water encapsulated within reverse micelles were investigated by solution NMR. Reverse micelles composed of AOT and pentane and solutions with varying concentrations of NaCl were studied at temperatures ranging from 20 degrees C to -30 degrees C. One-dimensional (1)H solution NMR spectroscopy was used to monitor the quantity and structure of encapsulated water. At low temperatures, e.g., -30 degrees C, reverse micelles lose water at rates that are dependent on the ionic strength of the aqueous nanopool. The final water loading (w0 = [water]/[surfactant]) of the reverse micelles is likewise dependent on the ionic strength of the aqueous phase. Remarkably, water resonance(s) at temperatures between -20 degrees C and -30 degrees C displayed fine structure indicating the presence of multiple transient water populations. Results of this study demonstrate that reverse micelles are an excellent vehicle for studies of confined water across a broad range of conditions, including the temperature range that provides access to the supercooled state.     

New reverse micelle surfactant systems optimized for high-resolution NMR spectroscopy of encapsulated proteins

Sodium bis(2-ethylhexyl)sulfosuccinate (AOT) is a surfactant commonly used to encapsulate water soluble proteins within the aqueous core of a reverse micelle. In the context of high-resolution NMR studies of encapsulated proteins the size of the resulting reverse micelle is critically important. We have designed and synthesized a short AOT analogue, 3,3-dimethyl-1-butylsulfosuccinate sodium salt and determined that it is able to form reverse micelles and to encapsulate the protein ubiquitin with high structural fidelity. AOT is often found to significantly destabilize encapsulated proteins, largely through charge-charge interactions between the anionic headgroup and the surface of the protein. Here we demonstrate, for the first time, that proportional mixtures of anionic and cationic surfactants can form reverse micelles that are also capable of protein encapsulation with high fidelity.     

Effects of temperature, salt, and deuterium oxide on the self-aggregation of alkylglycosides in dilute solution. 1. n-nonyl-beta-D-glucoside

The influence of salt, temperature, and deuterium oxide on the self-aggregation of n-nonyl-beta-D-glucoside (beta-C9G1) in dilute solution has been investigated by static and dynamic light scattering, neutron scattering, and tensiometry. Scattering data show that the micelles can be described as relatively stiff, elongated structures with a circular cross section. With a decrease of temperature, the micelles grow in one dimension, which makes it surprising that the critical micelle concentration (cmc) shows a concomitant increase. On the other hand, substitution of D2O for H2O causes a large increase in micelle size at low temperatures, without any appreciable effect on cmc. With increasing temperature, the deuterium effect on the micelle size diminishes. The effects of salt on the micelle size and cmc were found to follow the Hofmeister series. Thus, at constant salt concentration, the micelle size decreased according to the sequence SO4(2-) > Cl- > Br- > NO3- > I- > SCN-, whereas the effect on cmc displays the opposite trend. Here, I- and SCN are salting-in anions. Similarly, the effects of cations decrease with increasing polarizability in the sequence Li+ > Na+ > K+ > Cs+. At high ionic strength, the systems separate into two micellar phases. The results imply that the size of beta-C9G1 micelles is extremely sensitive to changes in the headgroup size. More specifically, temperature and salt effects on effective headgroup size, including intermolecular interactions and water ofhydration, are suggested to be more decisive for the micelle morphology than the corresponding effects on unimer solubility.     

Simple oxovanadates as multiparameter probes of reverse micelles

Using a wide range of different methods, researchers have found that the environment inside reverse micelles differs from bulk aqueous solution in many ways. Here, we present a new tool, a series of aqueous oxovanadium(V) reactions, to probe pH, viscosity, and ionic strength in the aqueous interior of reverse micelles. In addition to their potential as anionic probe analogues to phosphates, simple oxovanadium(V) compounds have equilibrium characteristics in aqueous media exquisitely sensitive to their environment. Therefore, the speciation of vanadate equilibria can be used as a parameter to characterize the intramicellar medium. Vanadate speciation is monitored through 51V NMR spectroscopy, which also yields information through chemical shifts and linewidths of spectral features. The speciation observed suggests that the relative acidity of a basic vanadate stock solution is slightly reduced in large, w0 >or= 12, reverse micelles, but that for smaller reverse micelles, speciation reflects the strong interaction of these negatively charged oxometalates with the reverse micelle and suggest an increased solution viscosity in the reverse micelles. This interpretation is obtained through different responses closely linked to the reverse micellar size and the specific conditions in the stock solutions used to form reverse micelles.     

Novel zwitterionic reverse micelles for encapsulation of proteins in low-viscosity media

Large proteins remain inaccessible to structural NMR studies because of their unfavorable relaxation properties. Their solubilization in the aqueous core of reverse micelles, in a low-viscosity medium, represents a promising approach, provided that their native tertiary structure is maintained. However, the use of classical ionic surfactants may lead to protein unfolding, due to strong electrostatic interactions between the polar head groups and the protein charges. To design reverse micelles in which these interactions are weakened, a new zwitterionic surfactant molecule was synthesized and studied by high-resolution NMR spectroscopy, for which cytochrome C and 15N-labeled ubiquitin were used as guest candidates. At different ionization states, both proteins are encapsulated in the absence of salts or other additives, in a folded conformation similar to the native one.     

Simulations of the confinement of ubiquitin in self-assembled reverse micelles

We describe the effects of confinement on the structure, hydration, and the internal dynamics of ubiquitin encapsulated in reverse micelles (RM). We performed molecular dynamics simulations of the encapsulation of ubiquitin into self-assembled protein/surfactant reverse micelles to study the positioning and interactions of the protein with the RM and found that ubiquitin binds to the RM interface at low salt concentrations. The same hydrophobic patch that is recognized by ubiquitin binding domains in vivo is found to make direct contact with the surfactant head groups, hydrophobic tails, and the iso-octane solvent. The fast backbone N-H relaxation dynamics show that the fluctuations of the protein encapsulated in the RM are reduced when compared to the protein in bulk. This reduction in fluctuations can be explained by the direct interactions of ubiquitin with the surfactant and by the reduced hydration environment within the RM. At high concentrations of excess salt, the protein does not bind strongly to the RM interface and the fast backbone dynamics are similar to that of the protein in bulk. Our simulations demonstrate that the confinement of protein can result in altered protein dynamics due to the interactions between the protein and the surfactant.     

Temperature dependence of the interaction of cholate and deoxycholate with fluid model membranes and their solubilization into mixed micelles

The interaction of bile salts with model membranes composed of soybean phosphatidylcholine (SPC) and synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated using high sensitivity isothermal titration calorimetry (ITC). The partitioning and incorporation of the bile salts sodium cholate (NaC) and sodium deoxycholate (NaDC) from an aqueous phase (pure water or 0.1 M NaCl) into fluid bilayer vesicles was studied as a function of temperature and ionic strength. The thermodynamic parameters of partitioning were determined with a model taking electrostatic interactions into account. In addition, the solubilization of SPC and POPC vesicles with NaC and NaDC as a function of temperature was also studied by ITC and the phase diagrams for the vesicle to micelle transition at two different temperatures were established. Unsaturated phospholipids require higher amounts of detergent to be transformed into micelles compared to saturated phospholipids. In addition, the width of the coexistence region of mixed micelles and mixed vesicles is larger for phosphatidylcholines with unsaturated chains. A comparison of NaDC with NaC shows the higher solubilization effectiveness of NaDC in agreement with its lower cmc. Furthermore, increasing the ionic strength decreases the amount of bile salt necessary for the formation of mixed micelles, which is also expected from the decrease of the cmc with ionic strength due to the shielding of the charges of the bile salts.     

High-resolution NMR studies of encapsulated proteins in liquid ethane

Many of the difficulties presented by large, aggregation-prone, and membrane proteins to modern solution NMR spectroscopy can be alleviated by actively seeking to increase the effective rate of molecular reorientation. An emerging approach involves encapsulating the protein of interest within the protective shell of a reverse micelle and dissolving the resulting particle in a low viscosity fluid, such as the short chain alkanes. Here we present the encapsulation of proteins with high structural fidelity within reverse micelles dissolved in liquid ethane. The addition of appropriate cosurfactants can significantly reduce the pressure required for successful encapsulation. At these reduced pressures, the viscosity of the ethane solution is low enough to provide sufficiently rapid molecular reorientation to significantly lengthen the spin-spin NMR relaxation times of the encapsulated protein.     

Protein Stability in Reverse Micelles

The N‐terminal SH3 domain of the Drosophila signal transduction protein drk was encapsulated in reverse micelles. Both the temperature of maximum stability and the melting temperature decreased on encapsulation. Dissecting the temperature‐dependent stability into enthalpic and entropic contributions reveals a stabilizing enthalpic and a destabilizing entropic contribution. These results do not match the expectations of hard‐core excluded volume theory, nor can they be wholly explained by interactions between the head groups in the reverse micelle and the test protein. We suggest that geometric constraints imposed by the reverse micelles need to be considered.     

Effects of temperature, salt, and deuterium oxide on the self-aggregation of alkylglycosides in dilute solution. 2. n-Tetradecyl-beta-D-maltoside

The effects of salt, temperature, and deuterium oxide on the self-aggregation of n-tetradecyl-beta-d-maltoside (C(14)G(2)) in dilute solution have been investigated by static light scattering, dynamic light scattering (DLS), small-angle neutron scattering (SANS), tensiometry, and capillary viscometry. SANS data show that the micelles can be described as relatively flexible polymer-like micelles with an elliptical cross section, at least at temperatures between 35 and 50 degrees C. The micelles grow in one dimension with increasing temperature and concentration. DLS and viscometry data suggest that the micelle size reaches a maximum at 60-70 degrees C. Comparison of DLS data in D(2)O and H(2)O shows that the micelles are larger in the former case. The effect of salt on the micelle size was found to follow the Hofmeister series. Thus, at constant salt concentration, the micelle size decreases according to the sequence SO(4)(2)(-) > Cl(-) > NO(3)(-) > I(-) > SCN(-), where I(-) and SCN(-) act as salting-in anions. From tensiometric data, it can be concluded that the temperature effects on micelle morphology do not correlate directly with those on unimer solubility. Rather, the temperature effect on the hydrocarbon chain conformation seems to be decisive for the micelle morphology. At constant temperature, on the other hand, the effect of salt and deuterium isotope is attributable to changes in effective headgroup area, including intermolecular interactions and water of hydration.     

Reverse micelles dissolved in supercritical xenon: an NMR spectroscopic study

Reverse micelles currently gain increasing interest in chemical technology. They also become important in biomolecular NMR due to their ability to host biomolecules such as proteins. In the present paper, a procedure for the preparation of high-pressure NMR samples containing reverse micelles dissolved in supercritical xenon is presented. These reverse micelles are formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT). For the first time, NMR spectroscopy could be applied to reverse micelles in supercritical xenon. The AOT/H(2)O/Xe system was studied as a function of experimental parameters such as xenon pressure, water content, and salt concentration. Optimum conditions for reverse micelle formation in supercritical xenon could be determined. It is, furthermore, demonstrated that biomolecules such as amino acids and proteins can be incorporated into the reverse micelles dissolved in supercritical xenon.     

Structure and interactions of charged triblock copolymers studied by small-angle X-ray scattering: dependence on temperature and charge screening

A series of thermo-responsive cationic triblock copolymers composed of methoxy-poly(ethylene glycol) (MPEG, hydrophilic), poly(N-isopropylacrylamide) (PNIPAAM, temperature sensitive), and poly((3-acrylamidopropyl) trimethyl ammonium chloride) (PN(+), cationic) has been investigated as a function of temperature and ionic strength. In the MPEG-b-PNIPAAM-b-PN(+) copolymers, the MPEG block length is constant, and the lengths of the PNIPAAM and PN(+) blocks are varied. The solubility of the PNIPAAM block decreases with increasing temperature, and the triblock copolymer thus provides the possibilities of studying micelles with both neutral and charged blocks in the micelle corona as well as the interplay between these two blocks as the electrostatic interactions are varied by addition of salt. Investigation of the systems by densitometry and small-angle X-ray scattering (SAXS) in a temperature range from 20 to 70 °C gave detailed information on the behavior both below and above the critical micelle temperature (CMT). A clear effect of the addition of salt is observed in both the apparent partial specific volume, obtained from the densitometry measurements, and the SAXS data. Below the CMT, the single polymers can be described as Gaussian chains, for which the repulsive interchain interactions, originating from the charged PN(+) block, have to be taken into account in salt-free aqueous solution. Increasing the salt concentration of the solution to 30 mM NaCl leads to an increase in the apparent partial specific volume, and the electrostatic repulsive interchain interactions between the single polymers vanish. Raising the temperature results in micelle formation, except for the copolymer with only 20 NIPAAM units. The SAXS data show that the polymer with the medium PNIPAAM block length forms spherical micelles, whereas the polymer with the longest PNIPAAM block forms cylindrical micelles. Increasing the temperature further above the CMT results in an increase in the micellar aggregation number for both of the polymers forming spherical and cylindrical micelles. The addition of salt to the solution also influences the aggregates formed above the CMT. Overall, the micelles formed in the salt solution have a smaller cross-section radius than those in aqueous solution without added salt.     

Evidence for micellar structure in the gas phase

We have compared micelles, reverse micelles, and reverse micelles encapsulating myoglobin using electrospray mass spectrometry. To enable a direct comparison, the same surfactant (cetyltrimethylammonium bromide (CTAB)) was used in each case and micelle formation was controlled by manipulating the aqueous and organic phases. Tandem mass spectra of the resulting micelle preparations reveal differences in the ions that dissociate: those that dissociate from regular micelles have undergone >90% exchange of bromide ions from the headgroup with acetate ions from bulk solvent. By contrast, for reverse micelles, ions are detected without exchange of bromide ions from the headgroup, consistent with their protection in the core of the micellar structure. Tandem mass spectra of micelles and reverse micelles reveal polydispersed assemblies containing several hundred CTAB molecules, indicating the coalescence of the micellar systems to form large assemblies. For reverse micelles incorporating myoglobin, spectra are consistent with one holo myogolobin molecule in association with approximately 270 CTAB molecules. Overall, therefore, our results show that the solution-phase orientation of surfactants is preserved during electrospray and are consistent with interactions being maintained between surfactants and an encapsulated protein.     

Extraction of Bovine Serum Albumin Using Reverse Micelles Formed by Hexadecyl Trimethyl Ammonium Chloride

The extraction of bovine serum albumin (BSA) has been investigated using reverse micelles of hexadecyl trimethyl ammonium chloride/n-octanol/isooctane. Forward extraction process parameters such as the surfactant concentration, co-solvent concentration, pH, ionic strength, and species of the initial aqueous phase were important factors affecting the extraction performance. These parameters were varied to optimize the extraction efficiency. Under the optimized conditions, forward extraction efficiencies of BSA can reach practically 99.55%. The thermodynamic study revealed that the extraction of BSA is controlled by entropy changes. Maximum back-extraction efficiency of 85.16% can be obtained at low pH values and high salt concentrations. The structures of BSA during reverse micelle extraction did not change by comparing the circular dichroism spectra of BSA back-extracted to the aqueous phase with that of feed BSA.     

Some Thoughts Regarding Theoretical Aspects of the Corrin-Harkins Relation and the Micellization Product of Ionic Micelles

The dependence of the stability of ionic micelles on the ionic strength of the medium is examined analytically without recourse to any explicit expression of the surface potential of micelles. The present study is based on the idea developed by Evans, Mitchell, and Ninham (D. F. Evans, D. J. Mitchell, and B. W. Ninham, J. Phys. Chem. 88, 6344 (1984)) that the interfacial free energy at the water/hydrocarbon core interface is independent of the ionic strength of the medium. The Corrin-Harkins (C-H) relation, a linear relation between the logarithm of the critical micelle concentration (cmc) and the logarithm of the counterion concentration n(C), is obtained in the range of n(C) where the salting-out effect is negligible, under the condition that the area per monomer on the micelle surface decreases very weakly with n(C). The "micellization product" of the charged pseudophase model of ionic micelles is discussed. The linear dependence of the surface potential of ionic micelles on n(C) is derived while a part of the effects of salt on the micelle size/shape is allowed. Copyright 2001 Academic Press.     

Surfactant-induced amorphous aggregation of tobacco mosaic virus coat protein: a physical methods approach

The interactions of non-ionic surfactant Triton X-100 and the coat protein of tobacco mosaic virus, which is an established model for both ordered and non-ordered protein aggregation, were studied using turbidimetry, differential scanning calorimetry, isothermal titration calorimetry, and dynamic light scattering. It was found that at the critical aggregation concentration (equal to critical micelle concentration) of 138 x 10(-6) M, Triton X-100 induces partial denaturation of tobacco mosaic virus coat protein molecules followed by protein amorphous aggregation. Protein aggregation has profound ionic strength dependence and proceeds due to hydrophobic sticking of surfactant-protein complexes (start aggregates) with initial radii of 46 nm. It has been suggested that the anionic surfactant sodium dodecyl sulfate forms mixed micelles with Triton X-100 and therefore reverses protein amorphous aggregation with release of protein molecules from the amorphous aggregates. A stoichiometric ratio of 5 was found for Triton X-100-sodium dodecyl sulfate interactions.     

Electrostatic interactions of charged dipolar proteins in reverse micelles

The electrostatic interactions in a reverse micelle containing a small-ionized protein are studied by Monte Carlo simulation. The electrostatic contribution to the potential of mean force of the protein in the reverse micelle is determined for a neutral protein, a uniformly charged protein, and a uniformly charged protein with a dipole moment. The effect of addition of a simple electrolyte is studied. While symmetrically distributed micellar charge exerts no force on enclosed ionic species, the protein is driven to the micellar wall due to interactions with simple ions. Protein binding to the inner wall of the micelle can be regulated by added salt. The presence of a dipole drives the protein further to the wall. These effects are studied for several proteins characterized by different charges and dipole moments. For a weakly charged protein with a strong dipole moment the contribution of dipolar interaction to the free energy can represent a major driving force for protein solubilization in the microemulsion.     

Endogenous growth of the population of reverse micelles

The coupling reaction between cetylbromide (CB) and trimethylamine (TMA) to yield the surfactant cetyltrimethylammonium bromide (CTAB) is studied in the system chloroform/isooctane (2/1,v/v)/water in which CTAB forms reverse micelles. This system affords an endogenous micelle population growth, i.e., an increase of the concentration of the micelles due to appearance of the surfactant in situ. The reaction is studied in the presence of preexisting CTAB reverse micelles. The rate of CTAB formation is measured by NMR spectroscopy, and the endogenous micelle population growth is directly monitored by time-resolved fluorescence quenching analysis. Under our experimental conditions, a 100% yield of the chemical reaction brings about a fourfold increase in the population of the reverse micelles. Since the water concentration is constant during chemical reaction, the newly formed water pools are formed at the expense of the initial ones, which brings about a decrease of the average water pool radius during micellar growth. The implication of the endogenous micelle population growth as a model for biological systems is briefly discussed.     

Solubilization properties of aqueous solutions of alkyltrimethylammonium halides toward a water-insoluble dye

The solubility of a water-insoluble dye, Sudan Red B, in aqueous sodium halide solutions of tetradecyl-, cetyl-, and stearyltrimethylammonium halides has been measured at different surfactant and salt concentrations, and the dependence of solubilization properties on alkyl chain length has been discussed with reference to the micelle size and shape. At low ionic strengths where only spherical micelles exist, the solubilization power of micellar surfactant slightly increases with increasing the ionic strength, but it sharply increases at high ionic strengths above the threshold value of sphere-rod transition. However, the solubilization power becomes independent of the ionic strength, if their rodlike micelles are sufficiently long. The solubilization capacity increases linearly with increasing the molecular weight, almost independent of counterion species, but the rod-like micelle has a higher solubilization capacity than the spherical micelle. The solubilization capacity is larger for a surfactant with longer alkyl chain, indicating that the dye is solubilized more readily in a larger hydrophobic core. The solubilized dye is situated in a rodlike micelle of alkyltrimethylammonium halides, on average, 4.5–7.5 nm apart from each other.