Low-temperature studies of encapsulated proteins

Water-soluble proteins encapsulated within reverse micelles may be studied under a variety of conditions, including low temperature and a wide range of buffer conditions. Direct high-resolution detection of information relating to protein folding intermediates and pathways can be monitored by low-temperature solution NMR. Ubiquitin encapsulated within AOT reverse micelles was studied using multidimensional multinuclear solution NMR to determine the relationship between protein structure, temperature, and ionic strength. Ubiquitin resonances were monitored by 15N HSQC NMR experiments at varying temperatures and salt concentrations. Our results indicate that the structure of the encapsulated protein at low temperature experiences perturbation arising from two major influences, which are reverse micelle-protein interactions and low-temperature effects (e.g., cold denaturation). These two effects are impossible to distinguish under conditions of low ionic strength. Elevated concentrations of nondenaturing salt solutions defeat the effects of reverse micelle-protein interactions and reveal low-temperature protein unfolding. High ionic strength shielding stabilizes the reverse micelle at low temperatures, which reduces the electrostatic interaction between the protein and reverse micelle surfaces, allowing the phenomenon of cold denaturation to be explored.     

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.     

Study of reverse micelles of di-isobutyl-phenoxy-ethoxy-ethyl-dimethyl-benzyl ammonium methacrylate in benzene by nuclear magnetic resonance spectroscopy

(1)H NMR spectroscopy was used to investigate the aggregation of the surfactant di-isobutyl-phenoxy-ethoxy-ethyl-dimethyl-benzyl ammonium methacrylate (Hyamine-M) in benzene. Adding water makes swollen reverse micelles (microemulsion droplets). The droplets also contain cadmium ions and the sodium salt of the methacrylic acid. The critical micelle concentration of Hyamine-M was determined by NMR to be 3.95 mM under the current conditions. Two-dimensional NMR NOESY spectra were used to study the conformation of the surfactant in the micelle and the spatial localization of water and counterions. We found that the surfactant molecules are folded with both phenyl fragments oriented toward the micelle exterior and the oxyethylene and NCH(3) groups in the micelle core. The water molecules and counterions are distributed around the surfactant polar groups in the micelle interior and penetrate up to both aromatic rings. The investigated system can be further utilized as a microemulsion matrix for the synthesis of cadmium-containing semiconductor nanocrystals, eventually capped with a polymer shell, or of polymer nanoparticles.     

Probing of ferrocenylanilines on model micelle/reverse micelle membrane and their enhanced reactivity for reactive oxidants

Reactive oxygen species are formed in the human body but can be removed by suitable antioxidants. In this study we synthesized and characterized three ferrocene derivatives, 4‐ferrocenylaniline (pFA), 3‐ferrocenylaniline (mFA) and 3‐methyl‐4‐ferrocenylaniline (MeFA), having significant potential to be used as antioxidants. The synthesized compounds are insoluble in water, with the solubility of these compounds increasing in micelle solution. The micelle and reverse micelle solutions were considered as model membranes. The synthesized compounds were probed on the model membranes, made by sodium dioctylsulfosuccinate reverse micelle and tetradecyltrimethylammonium bromide micelle, using ¹H NMR spectroscopy. The ¹H NMR results indicated that these compounds are present in the polar region of the model membrane interface. Quantitative measurements showed that mFA has the greatest ability to penetrate into the micelle membrane among these compounds, and pFA is least penetrating in this respect. Solubilization of these compounds in aqueous micelle solution facilitates crystallization (of mFA) and enhances the antioxidant potential of these compounds. X‐ray crystal structure analysis revealed that mFA captures water molecules during crystallization in micelle solution. Their ability to act as antioxidants was evaluated, in dimethylsulfoxide (DMSO) and in micelle solution, using standard 1,1‐ diphenyl‐2‐picrylhydrazyl (DPPH) assay. It was found that their antioxidant potential is good in DMSO and that potential increases on the interface of the model membrane. The highest increase (by 19.6%) in the antioxidant potential, on the model membrane interface, was observed for mFA.     

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.     

Pressure-induced protein unfolding in the ternary system AOT-octane-water is different from that in bulk water

In a cellular environment, the presence of macromolecular cosolutes and membrane interfaces can influence the folding-unfolding behavior of proteins. Here we report on the pressure stability of alpha-chymotrypsin in the ternary system bis(2-ethylhexyl)sodium sulfosuccinate-octane-water using FTIR spectroscopy. The ternary system forms anionic reverse micelles which mimic cellular conditions. We find that inclusion of a single protein molecule in a reverse micelle does not alter its conformation. When pressurized in bulk water, alpha-chymotrypsin unfolds at 750 MPa into a partially unfolded structure. In contrast, in the ternary system, the same pressure increase induces a random coil-like unfolded state, which collapses into an amorphous aggregate during the decompression phase. It is suggested that the unfolding pathway is different in a cell-mimicking environment due to the combined effect of multiple factors, including confinement. A phase transition of the reverse micellar to the lamellar phase is thought to be essential to provide the conditions required for unfolding and aggregation, though the unfolding is not a direct result of the phase transition. Our observations therefore suggest that membranes may cause the formation of alternative conformations that are more susceptible to aggregation.     

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.     

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.     

Unfolding and refolding details of lysozyme in the presence of β-casein micelles

In this work, we selected a small globular protein, lysozyme, to study how it unfolds and refolds in the presence of micelles composed of the unstructured β-casein proteins by using microcalorimetry and circular dichroism spectroscopy. It was found that a partially unfolded structure of lysozyme starts to form when the β-casein/lysozyme molar ratio is above 0.7, and the structure forms exclusively when the β-casein/lysozyme molar ratio is above 1.6. This partially unfolded state of lysozyme loses most of its tertiary structure and after heating, the denatured lysozyme molecules are trapped in the charged coatings of β-casein micelles and cannot refold upon cooling. The thus obtained protein complex can be viewed as a kind of special polyelectrolyte complex micelle. The net charge ratios of the two proteins and the ionic strength of the dispersions can significantly modulate the electrostatic and hydrophobic interactions between the two proteins. Our present work may have implications for the nanoparticle protein engineering therapy in the biomedicine field and may provide a better understanding of the principles governing the protein-protein interactions. Besides, the heating-cooling-reheating procedure employed in this work can also be used to study the unfolding and refolding details of the target protein in other protein-protein, protein-polymer and protein-small solute systems.     

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.     

Mapping the hydration dynamics of ubiquitin

The nature of water's interaction with biomolecules such as proteins has been difficult to examine in detail at atomic resolution. Solution NMR spectroscopy is potentially a powerful method for characterizing both the structural and temporal aspects of protein hydration but has been plagued by artifacts. Encapsulation of the protein of interest within the aqueous core of a reverse micelle particle results in a general slowing of water dynamics, significant reduction in hydrogen exchange chemistry and elimination of contributions from bulk water thereby enabling the use of nuclear Overhauser effects to quantify interactions between the protein surface and hydration water. Here we extend this approach to allow use of dipolar interactions between hydration water and hydrogens bonded to protein carbon atoms. By manipulating the molecular reorientation time of the reverse micelle particle through use of low viscosity liquid propane, the T(1ρ) relaxation time constants of (1)H bonded to (13)C were sufficiently lengthened to allow high quality rotating frame nuclear Overhauser effects to be obtained. These data supplement previous results obtained from dipolar interactions between the protein and hydrogens bonded to nitrogen and in aggregate cover the majority of the molecular surface of the protein. A wide range of hydration dynamics is observed. Clustering of hydration dynamics on the molecular surface is also seen. Regions of long-lived hydration water correspond with regions of the protein that participate in molecular recognition of binding partners suggesting that the contribution of the solvent entropy to the entropy of binding has been maximized through evolution.     

Residue-specific real-time NMR diffusion experiments define the association states of proteins during folding

Characterizing the association states of proteins during folding is critical for understanding the nature of protein-folding intermediates and protein-folding pathways, protein aggregation, and disease-related aggregation. To study the association states of unfolded, folded, and intermediate species during protein folding, we have introduced a novel residue-specific real-time NMR diffusion experiment. This experiment, a combination of NMR real-time folding experiments and 3D heteronuclear pulsed field gradient NMR diffusion experiments (LED-HSQC), measures hydrodynamic properties, or molecular sizes, of kinetic species directly during the folding process. Application of the residue-specific real-time NMR diffusion experiments to characterize the folding of the collagen triple helix motif shows that this experiment can be used to determine the association states of unfolded, folded, and kinetic intermediates with transient lifetimes simultaneously. The ratio of the apparent translational diffusion coefficients of the unfolded to the folded form of the triple helix is 0.59, which correlates very well with a theoretical ratio for monomer to linear trimer. The apparent diffusion coefficients of the kinetic intermediates formed during triple helix folding indicate the formation of trimer-like associates which is consistent with previously published kinetic and relaxation data. The residue-specific time dependence of apparent diffusion coefficients of monomer and trimer peaks also illustrates the ability to use diffusion data to probe the directionality of triple helix formation. NMR diffusion experiments provide a new strategy for the investigation of protein-folding mechanisms, both to understand the role of kinetic intermediates and to determine the time-dependent aggregation processes in human diseases.     

An example of how to use AOT reverse micelle interfaces to control a photoinduced intramolecular charge-transfer process

6-Propionyl-2-(N,N-dimethyl)aminonaphtahalene, PRODAN, is widely used as a fluorescent molecular probe due to its significant Stokes shift in polar solvents. It is an aromatic compound with intramolecular charge-transfer (ICT) states which can be particularly useful as sensors. In this work, we performed absorption, steady-state, time-resolved fluorescence (TRES), and time-resolved area normalized emission (TRANES) spectroscopies on PRODAN dissolved in nonaqueous reverse micelles. The reverse micelles are composed of polar solvents/sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane. Sequestered polar solvents included ethylene glycol (EG), propylene glycol (PG), glycerol (GY), formamide (FA), dimethylformamide (DMF), and dimethylacetamide (DMA). The experiments were performed with varying surfactant concentrations at a fixed molar ratio W(S) = [polar solvent]/[AOT]. In every reverse micelle studied, the results show that PRODAN undergoes a partition process between the external solvent and the reverse micelle interface. The partition constants, K(p), are quantified from the changes in the PRODAN emission and/or absorption spectra with the surfactant concentration. The K(p) values depend strongly on the encapsulated polar solvent and correlate quite well with the AOT reverse micelle interface's zones where PRODAN can exist and emits. Thus, the partition toward the reverse micelle interface is strongly favored in DMF and DMA containing micelles where the PRODAN emission comes only from an ICT state. For GY/AOT reverse micelles, the K(p) value is the lowest and only emission from the local excited (LE) state is observed. On the other hand, for EG/AOT, PG/AOT, and water/AOT reverse micelles, the K(p) values are practically the same and emission from both states (LE and ICT) is simultaneously detected. We show here that it is possible to control the PRODAN state emission by simply changing the properties of the AOT reverse micelle interfaces by choosing the appropriate polar solvent to make the reverse micelle media. Indeed, we present experimental evidence with the answer to the long time question about from which state does PRODAN emit, a process that can be controlled using the unique reverse micelle interfaces properties.     

Macromolecular crowding fails to fold a globular protein in cells

Proteins perform their functions in cells where macromolecular solutes reach concentrations of >300 g/L and occupy >30% of the volume. The volume excluded by these macromolecules stabilizes globular proteins because the native state occupies less space than the denatured state. Theory predicts that crowding can increase the ratio of folded to unfolded protein by a factor of 100, amounting to 3 kcal/mol of stabilization at room temperature. We tested the idea that volume exclusion dominates the crowding effect in cells using a variant of protein L, a 7 kDa globular protein with seven lysine residues replaced by glutamic acids; 84% of the variant molecules populate the denatured state in dilute buffer at room temperature, compared with 0.1% for the wild-type protein. We then used in-cell NMR spectroscopy to show that the cytoplasm of Escherichia coli does not overcome even this modest (～1 kcal/mol) free-energy deficit. The data are consistent with the idea that nonspecific interactions between cytoplasmic components can overcome the excluded-volume effect. Evidence for these interactions is provided by the observations that adding simple salts folds the variant in dilute solution but increasing the salt concentration inside E. coli does not fold the protein. Our data are consistent with the results of other studies of protein stability in cells and suggest that stabilizing excluded-volume effects, which must be present under crowded conditions, can be ameliorated by nonspecific interactions between cytoplasmic components.     

Protein folding in a reverse micelle environment: the role of confinement and dehydration

Characterization of the molecular interactions that stabilize the folded state of proteins including hydrogen bond formation, solvation, molecular crowding, and interaction with membrane environments is a fundamental goal of theoretical biophysics. Inspired by recent experimental studies by Gai and co-workers, we have used molecular dynamics simulations to explore the structure and dynamics of the alanine-rich AKA(2) peptide in bulk solution and in a reverse micelle environment. The simulated structure of the reverse micelle shows substantial deviations from a spherical geometry. The AKA(2) peptide is observed to (1) remain in a helical conformation within a spherically constrained reverse micelle and (2) partially unfold when simulated in an unconstrained reverse micelle environment, in agreement with experiment. While aqueous solvation is found to stabilize the N- and C-termini random coil portions of the peptide, the helical core region is stabilized by significant interaction between the nonpolar surface of the helix and the aliphatic chains of the AOT surfactant. The results suggest an important role for nonpolar peptide-surfactant and peptide-lipid interactions in stabilizing helical geometries of peptides in reverse micelle environments.     

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.     

Correlating proton transfer dynamics to probe location in confined environments

The dramatic impact of differing environments on proton transfer dynamics of the photoacid HPTS prompted us to investigate these systems with two highly complementary methods: ultrafast time-resolved transient absorption and two-dimensional NMR spectroscopies. Both ultrafast time-resolved transient absorption spectroscopy and time-resolved anisotropy decays demonstrate the proton transfer dynamics depend intimately on the specific reverse micellar system. For w(0) = 10 reverse micelles formed with anionic AOT surfactant, the HPTS proton transfer dynamics are similar to dynamics in bulk aqueous solution, and the corresponding (1)H 2D NOESY NMR spectra display no cross peaks between HPTS and AOT consistent with the HPTS residing well hydrated by water in the interior of the reverse micelle water pool. In contrast, ultrafast transient absorption experiments show no evidence for HPTS photoinduced proton transfer reaction in reverse micelles formed with the cationic CTAB surfactant. In CTAB reverse micelles, clear cross peaks between HPTS and CTAB in the 2D NMR spectra show that HPTS embeds in the interface. These results indicate that the environment strongly impacts the proton transfer reaction and that complementary experimental techniques develop understanding of how location critically affects molecular responses.     

Characterization of folding intermediates of a domain-swapped protein by solid-state NMR spectroscopy

We have employed two-dimensional solid-state NMR to study structure and dynamics of insoluble folding states of the domain-swapped protein Crh. Starting from the protein precipitated at its pI, conformational changes due to a modest temperature increase were investigated at the level of individual residues and in real-time. As compared to the crystalline state, Crh pI-precipitates exhibited a higher degree of molecular mobility for several regions of the protein. A rigidly intact center was observed including a subset of residues of the hydrophobic core. Raising the temperature by 13 K to 282 K created a partially unfolded intermediate state that was converted into beta-sheet-rich aggregates that are mostly of spherical character according to electron microscopy. Residue-by-residue analysis indicated that two out of three alpha-helices in aggregated Crh underwent major structural rearrangements while the third helix was preserved. Residues in the hinge region exhibited major chemical-shift changes, indicating that the domain swap was not conserved in the aggregated form. Our study provides direct evidence that protein aggregates of a domain-swapped protein retain a significant fraction of native secondary structure and demonstrates that solid-state NMR can be used to directly monitor slow molecular folding events.