Preparation,characterization, and NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids

Encapsulating a protein in a reverse micelle and dissolving it in a low-viscosity solvent can lower the rotational correlation time of a protein and thereby provides a novel strategy for studying proteins in a variety of contexts. The preparation of the sample is a key element in this approach and is guided by a number of competing parameters. Here we examine the applicability of several strategies for the preparation and characterization of encapsulated proteins dissolved in low viscosity fluids that are suitable for high performance NMR spectroscopy. Ubiquitin is used as a model system to explore various issues such as the homogeneity of the encapsulation, characterization of the hydrodynamic performance of reverse micelles containing protein molecules, and the effective pH of the water environment of the reverse micelle.     

A method for solution NMR structural studies of large integral membrane proteins: Reverse micelle encapsulation

The structural study of membrane proteins perhaps represents one of the greatest challenges of the post-genomic era. While membrane proteins comprise over 50% of current and potential drug targets, their structural characterization lags far behind that of soluble proteins. Nuclear magnetic resonance (NMR) offers great potential not only with respect to structural characterization of integral membrane proteins but may also provide the ability to study the details of small ligand interactions. However, the size limitations of solution NMR have restricted comprehensive structural characterization of membrane protein NMR structures to the relatively small β-barrel proteins or helical proteins of relatively simple topology. In an effort to escape the barriers presented by slow molecular reorientation of large integral membrane proteins solubilized by detergent micelles in water, we have adapted the reverse micelle encapsulation strategy originally developed for the study of large soluble proteins by solution NMR methods. Here we review a novel approach to the solubilization of large integral membrane proteins in reverse micelle surfactants dissolved in low viscosity alkane solvents. The procedure is illustrated with a 54 kDa construct of the homotetrameric KcsA potassium channel.     

Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids

Comprehensive application of solution NMR spectroscopy to studies of macromolecules remains fundamentally limited by the molecular rotational correlation time. For proteins, molecules larger than 30 kDa require complex experimental methods, such as TROSY in conjunction with isotopic labeling schemes that are often expensive and generally reduce the potential information available. We have developed the reverse micelle encapsulation strategy as an alternative approach. Encapsulation of proteins within the protective nano-scale water pool of a reverse micelle dissolved in ultra-low viscosity nonpolar solvents overcomes the slow tumbling problem presented by large proteins. Here, we characterize the contributions from the various components of the protein-containing reverse micelle system to the rotational correlation time of the encapsulated protein. Importantly, we demonstrate that the protein encapsulated in the reverse micelle maintains a hydration shell comparable in size to that seen in bulk solution. Using moderate pressures, encapsulation in ultra-low viscosity propane or ethane can be used to magnify this advantage. We show that encapsulation in liquid ethane can be used to reduce the tumbling time of the 43 kDa maltose binding protein from ~23 to ~10 ns. These conditions enable, for example, acquisition of TOCSY-type data resolved on the adjacent amide NH for the 43 kDa encapsulated maltose binding protein dissolved in liquid ethane, which is typically impossible for proteins of such size without use of extensive deuteration or the TROSY effect.     

Novel surfactant mixtures for NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids

NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids is emerging as a tool for biophysical studies of proteins in atomic detail in a variety of otherwise inaccessible contexts. The central element of the approach is the encapsulation of the protein of interest within the aqueous core of a reverse micelle with high structural fidelity. The process of encapsulation is highly dependent upon the nature of the surfactant(s) employed. Here we describe novel mixtures of surfactants that are capable of successfully encapsulating a range of types of proteins under a variety of conditions.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : III. VISCOSITY.

1. Gelatin solutions have a high viscosity which in the case of freshly prepared solutions varies under the influence of the hydrogen ion concentration in a similar way as the swelling, the osmotic pressure, and the electromotive forces. Solutions of crystalline egg albumin have under the same conditions a comparatively low viscosity which is practically independent of the pH (above 1.0). This difference in the viscosities of solutions of the two proteins seems to be connected with the fact that solutions of gelatin have a tendency to set to a Jelly while solutions of crystalline egg albumin show no such tendency at low temperature and pH above 1.0. 2. The formulæ for viscosity demand that the difference in the order of magnitude of the viscosity of the two proteins should correspond to a difference in the relative volume occupied by equal masses of the two proteins in the same volume of solution. It is generally assumed that these variations of volume of dissolved proteins are due to the hydration of the isolated protein ions, but if this view were correct the influence of pH on viscosity should be the same in the case of solutions of gelatin, of amino-acids, and of crystalline egg albumin, which, however, is not true. 3. Suspensions of powdered gelatin in water were prepared and it was found, first, that the viscosity of these suspensions is a little higher than that of gelatin solutions of the same concentration, second, that the pH influences the viscosity of these suspensions similarly as the viscosity of freshly prepared gelatin solutions, and third, that the volume occupied by the gelatin in the suspension varies similarly as the viscosity which agrees with the theories of viscosity. It is shown that this influence of the pH on the volume occupied by the gelatin granules in suspension is due to the existence of a Donnan equilibrium between the granules and the surrounding solution.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : IV. VISCOSITY—Continued.

1. The proof is completed that the influence of electrolytes on the viscosity of suspensions of powdered particles of gelatin in water is similar to the influence of electrolytes on the viscosity of solutions of gelatin in water. 2. It has been suggested that the high viscosity of proteins is due to the existence of a different type of viscosity from that existing in crystalloids. It is shown that such an assumption is unnecessary and that the high viscosity of solutions of isoelectric gelatin can be accounted for quantitatively on the assumption that the relative volume of the gelatin in solution is comparatively high. 3. Since isoelectric gelatin is not ionized, the large volume cannot be due to a hydration of gelatin ions. It is suggested that this high volume of gelatin solutions is caused by the existence in the gelatin solution of submicroscopic pieces of solid gelatin occluding water, the relative quantity of which is regulated by the Donnan equilibrium. This would also explain why the influence of electrolytes on the viscosity of gelatin solutions is similar to the influence of electrolytes on the viscosity of suspensions of particles of gelatin. 4. This idea is supported by experiments on solutions and suspensions of casein chloride in which it is shown that their viscosity is chiefly due to the swelling of solid particles of casein, occluding quantities of water regulated by the Donnan equilibrium; and that the breaking up of these solid particles into smaller particles, no longer capable of swelling, diminishes the viscosity. 5. This leads to the idea that proteins form true solutions in water which in certain cases, however, contain, side by side with isolated ions and molecules, submicroscopic solid particles capable of occluding water whereby the relative volume and the viscosity of the solution is considerably increased. This accounts not only for the high order of magnitude of the viscosity of such protein solutions but also for the fact that the viscosity is influenced by electrolytes in a similar way as is the swelling of protein particles. 6. We therefore reach the conclusion that there are two sources for the viscosity of protein solutions; one due to the isolated protein ions and molecules, and the other to the submicroscopic solid particles contained in the solution. The viscosity due to the isolated molecules and ions of proteins we will call the general viscosity since it is of a similar low order of magnitude as that of crystalloids in solution; while the high viscosity due to the submicroscopic solid protein particles capable of occluding water and of swelling we will call the special viscosity of protein solutions. Under ordinary conditions of hydrogen ion concentration and temperature (and in not too high a concentration of the protein in solution) the general viscosity due to isolated ions and molecules prevails in solutions of crystalline egg albumin and in solutions of metal caseinates (where the metal is monovalent) while under the same conditions the second type of viscosity prevails in solutions of gelatin and in solutions of acid-salts of casein; and also in solutions of crystalline egg albumin at a pH below 1.0 and at higher temperatures. The special viscosity is higher in solutions of gelatin than of casein salts for the probable reason that the amount of water occluded by the submicroscopic solid gel particles in a gelatin solution is, as a rule, considerably higher than that occluded by the corresponding particles of casein.     

Exploring weak, transient protein--protein interactions in crowded in vivo environments by in-cell nuclear magnetic resonance spectroscopy

Biology relies on functional interplay of proteins in the crowded and heterogeneous environment inside cells, and functional protein interactions are often weak and transient. Thus, methods that preserve these interactions and provide information about them are needed. In-cell nuclear magnetic resonance (NMR) spectroscopy is an attractive method for studying a protein's behavior in cells because it may provide residue-level structural and dynamic information, yet several factors limit the feasibility of protein NMR spectroscopy in cells; among them, slow rotational diffusion has emerged as the most important. In this paper, we seek to elucidate the causes of the dramatically slow protein tumbling in cells and in so doing to gain insight into how the intracellular viscosity and weak, transient interactions modulate protein mobility. To address these questions, we characterized the rotational diffusion of three model globular proteins in Escherichia coli cells using two-dimensional heteronuclear NMR spectroscopy. These proteins have a similar molecular size and globular fold but very different surface properties, and indeed, they show very different rotational diffusion in the E. coli intracellular environment. Our data are consistent with an intracellular viscosity approximately 8 times that of water, too low to be a limiting factor for observation of small globular proteins by in-cell NMR spectroscopy. Thus, we conclude that transient interactions with cytoplasmic components significantly and differentially affect the mobility of proteins and therefore their NMR detectability. Moreover, we suggest that an intricate interplay of total protein charge and hydrophobic interactions plays a key role in regulating these weak intermolecular interactions in cells.     

Peptides and proteins in a confined environment: NMR spectra at natural isotopic abundance.

Confinement of proteins and peptides in a small inert space mimics the natural environment of the cell, allowing structural studies in conditions that stabilize folded conformations. We have previously shown that confinement in polyacrylamide gels (PAGs) is sufficient to induce a change in the viscosity of the aqueous solution without changing the composition and temperature of the solvent. The main limitation of a PAG to run NMR experiments in a confined environment is the need for labelling the peptides. Here we report the use of the agarose gel to run the NMR spectra of proteins and peptides. We show that agarose gels are completely transparent in NMR experiments, relieving the need for labelling. Although it is necessary to expose biomolecules to fairly high temperatures during sample preparation, we believe that this is not generally an obstacle to the study of peptides, and found that the method is also compatible with temperature-resistant proteins. The mesh of agarose gels is too wide for direct effects of confinement on the stability of proteins but confinement can be easily exploited to interact the proteins with other reagents, including crowding macromolecules that can eventually lead to fold stabilization. The use of these gels is ideally suited for low-temperature studies; we show that a very flexible peptide at subzero temperatures is stabilized into a well-folded conformation.     

Rapid assignment of solution 31P NMR spectra of large proteins by solid-state spectroscopy

The application of the (31)P NMR spectroscopy to large proteins or protein complexes in solution is hampered by a relatively low intrinsic sensitivity coupled with large line widths. Therefore, the assignment of the phosphorus signals by two-dimensional NMR methods in solution is often extremely time consuming. In contrast, the quality of solid-state NMR spectra is not dependent on the molecular mass and the solubility of the protein. For the complex of Ras with the GTP-analogue GppCH(2)p we show solid-state (31)P NMR methods to be more sensitive by almost one order of magnitude than liquid-state NMR. Thus, solid-state NMR seems to be the method of choice for obtaining the resonance assignment of the phosphorus signals of protein complexes in solution. Experiments on Ras.GDP complexes show that the microcrystalline sample can be substituted by a precipitate of the sample and that unexpectedly the two structural states observed earlier in solution are present in crystals as well.     

Characterizing monoclonal antibody formulations in arginine glutamate solutions using 1H NMR spectroscopy

Assessing how excipients affect the self-association of monoclonal antibodies (mAbs) requires informative and direct in situ measurements for highly concentrated solutions, without sample dilution or perturbation. This study explores the application of solution nuclear magnetic resonance (NMR) spectroscopy for characterization of typical mAb behavior in formulations containing arginine glutamate. The data show that the analysis of signal intensities in 1D ¹H NMR spectra, when compensated for changes in buffer viscosity, is invaluable for identifying conditions where protein-protein interactions are minimized. NMR-derived molecular translational diffusion rates for concentrated solutions are less useful than transverse relaxation rates as parameters defining optimal formulation. Furthermore, NMR reports on the solution viscosity and mAb aggregation during accelerated stability study assessment, generating data consistent with that acquired by size-exclusion chromatography. The methodology developed here offers NMR spectroscopy as a new tool providing complementary information useful to formulation development of mAbs and other large therapeutic proteins.     

NMR structures of paramagnetic metalloproteins

Metalloproteins represent a large share of the proteome and many of them contain paramagnetic metal ions. The knowledge, at atomic resolution, of their structure in solution is important to understand processes in which they are involved, such as electron transfer mechanisms, enzymatic reactions, metal homeostasis and metal trafficking, as well as interactions with their partners. Formerly considered as unfeasible, the first structure in solution by nuclear magnetic resonance (NMR) of a paramagnetic protein was obtained in 1994. Methodological and instrumental advancements pursued over the last decade are such that NMR structure of paramagnetic proteins may be now routinely obtained. We focus here on approaches and problems related to the structure determination of paramagnetic proteins in solution through NMR spectroscopy. After a survey of the background theory, we show how the effects produced by the presence of a paramagnetic metal ion on the NMR parameters, which are in many cases deleterious for the detection of NMR spectra, can be overcome and turned into an additional source of structural restraints. We also briefly address features and perspectives given by the use of 13C-detected protonless NMR spectroscopy for proteins in solution. The structural information obtained through the exploitation of a paramagnetic center are discussed for some Cu2+ -binding proteins and for Ca2+ -binding proteins, where the replacement of a diamagnetic metal ion with suitable paramagnetic metal ions suggests novel approaches to the structural characterization of proteins containing diamagnetic and NMR-silent metal ions.     

1H NMR of compounds with low water solubility in the presence of erythrocytes: effects of emulsion phase separation

When lipophilic compounds like diethyl phthalate (DEP) were added to water, two sets of resonances appeared in the 1H NMR spectrum, whereas when added in concentrations above approximately 3.5 mM to erythrocytes in a high haematocrit suspension, only one set of resonances was observed at the low-frequency position. The appearance of one set of resonances at lower frequency was found to be common to a series of lipophilic compounds in erythrocytes. The appearance of the NMR spectra is ascribed to the existence of an emulsion, meaning two different phases of a compound: a "droplet" (resonances to lower frequency) and aqueous dissolved phase (resonances to higher frequency). The absence of the resonances from the dissolved phase in erythrocyte solution is ascribed to exchange broadening. The absolute chemical shift of the compound in its "droplet" phase was also measured using a cylindrical/spherical microcell. This arrangement mimicked the geometry of the dissolved versus the phase-separated species and thus obviated the effect of a difference in magnetic susceptibility between the "droplet" solute and its aqueous solution. Factors influencing the formation of emulsion phases such as erythrocytes, haemoglobin and smaller proteins were investigated; they are found to be effective in the order given.     

The spreading of monolayers of myosin

The spreading of the muscle proteins myosin and actomyosin depends both on the salt solution in which the proteins are dissolved and on the solution on which they are spread. The spreading is more complete the lower the concentration of the salt solution in which the proteins are dissolved and the higher the salt concentration of the solutions on which the proteins are spread. This effect seems to be due partially to the difference in density allowing the spread droplets a longer life on the surface, and partially to the effect of salt on the charge of the protein. A change in the pH of the substrate has a smaller effect than a change in the salt concentration. Heavy metals like Cu⁺⁺ or Zn⁺⁺, inhibit spreading almost completely. The dependence of spreading on these salts and on time was investigated in detail.The effect of the different salts was also compared with the effect of different phosphate compounds. It was noted that the above substances, including the different salts, phosphate compounds, and heavy metals, affect the mechanism of spreading but not films already spread. The viscosity of these fibrillar proteins was compared with other proteins in the monomolecular film state and in myosin an unusually high viscosity was found.     

Use of reverse micelles in membrane protein structural biology

Membrane protein structural biology is a rapidly developing field with fundamental importance for elucidating key biological and biophysical processes including signal transduction, intercellular communication, and cellular transport. In addition to the intrinsic interest in this area of research, structural studies of membrane proteins have direct significance on the development of therapeutics that impact human health in diverse and important ways. In this article we demonstrate the potential of investigating the structure of membrane proteins using the reverse micelle forming surfactant dioctyl sulfosuccinate (AOT) in application to the prototypical model ion channel gramicidin A. Reverse micelles are surfactant based nanoparticles which have been employed to investigate fundamental physical properties of biomolecules. The results of this solution NMR based study indicate that the AOT reverse micelle system is capable of refolding and stabilizing relatively high concentrations of the native conformation of gramicidin A. Importantly, pulsed-field-gradient NMR diffusion and NOESY experiments reveal stable gramicidin A homodimer interactions that bridge reverse micelle particles. The spectroscopic benefit of reverse micelle-membrane protein solubilization is also explored, and significant enhancement over commonly used micelle based mimetic systems is demonstrated. These results establish the effectiveness of reverse micelle based studies of membrane proteins, and illustrate that membrane proteins solubilized by reverse micelles are compatible with high resolution solution NMR techniques. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mass spectrometric approaches using electrospray ionization charge states and hydrogen-deuterium exchange for determining protein structures and their conformational changes

Electrospray ionization (ESI) mass spectrometry (MS) is a powerful analytical tool for elucidating structural details of proteins in solution especially when coupled with amide hydrogen/deuterium (H/D) exchange analysis. ESI charge-state distributions and the envelopes of charges they form from proteins can provide an abundance of information on solution conformations that is not readily available through other biophysical techniques such as near ultraviolet circular dichroism (CD) and tryptophan fluorescence. The most compelling reason for the use of ESI-MS over nuclear magnetic resonance (NMR) for measuring H/D after exchange is that larger proteins and lesser amounts of samples can be studied. In addition, MS can provide structural details on transient or folding intermediates that may not be accessible by CD, fluorescence, and NMR because these techniques measure the average properties of large populations of proteins in solution. Correlations between measured H/D and calculated parameters that are often available from crystallographic data can be used to extend the range of structural details obtained on proteins. Molecular dynamics and energy minimization by simulation techniques such as assisted model building with energy refinement (AMBER) force field can be very useful in providing structural models of proteins that rationalize the experimental H/D exchange results. Charge-state envelopes and H/D exchange information from ESI-MS data used complementarily with NMR and CD data provides the most powerful approach available to understanding the structures and dynamics of proteins in solution.     

Choosing membrane mimetics for NMR structural studies of transmembrane proteins

The native environment of membrane proteins is complex and scientists have felt the need to simplify it to reduce the number of varying parameters. However, experimental problems can also arise from oversimplification which contributes to why membrane proteins are under-represented in the protein structure databank and why they were difficult to study by nuclear magnetic resonance (NMR) spectroscopy. Technological progress now allows dealing with more complex models and, in the context of NMR studies, an incredibly large number of membrane mimetics options are available. This review provides a guide to the selection of the appropriate model membrane system for membrane protein study by NMR, depending on the protein and on the type of information that is looked for. Beside bilayers (of various shapes, sizes and lamellarity), bicelles (aligned or isotropic) and detergent micelles, this review will also describe the most recent membrane mimetics such as amphipols, nanodiscs and reverse micelles. Solution and solid-state NMR will be covered as well as more exotic techniques such as DNP and MAOSS.     

一种改进型体液表观粘度函数测量仪

本文介绍一种性能较好的体液表观粘度函数快测系统,它是在较严密的流变学理论基础上通过较精细的硬、软件设计而研究成功的.其前身为L-1型粘度计,但作了重要改进.它的最大特点是能在一次测量过程完后得到不同切变率下的表观粘度;同时能在通常认为困难却十分重要的“低剪切”段令人满意地工作.     

Refined solution structure of the 82-kDa enzyme malate synthase G from joint NMR and synchrotron SAXS restraints

Determination of the accurate three-dimensional structure of large proteins by NMR remains challenging due to a loss in the density of experimental restraints resulting from the often prerequisite perdeuteration. Solution small-angle scattering, which carries long-range translational information, presents an opportunity to enhance the structural accuracy of derived models when used in combination with global orientational NMR restraints such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs). We have quantified the improvements in accuracy that can be obtained using this strategy for the 82 kDa enzyme Malate Synthase G (MSG), currently the largest single chain protein solved by solution NMR. Joint refinement against NMR and scattering data leads to an improvement in structural accuracy as evidenced by a decrease from approximately 4.5 to approximately 3.3 A of the backbone rmsd between the derived model and the high-resolution X-ray structure, PDB code 1D8C. This improvement results primarily from medium-angle scattering data, which encode the overall molecular shape, rather than the lowest angle data that principally determine the radius of gyration and the maximum particle dimension. The effect of the higher angle data, which are dominated by internal density fluctuations, while beneficial, is also found to be relatively small. Our results demonstrate that joint NMR/SAXS refinement can yield significantly improved accuracy in solution structure determination and will be especially well suited for the study of systems with limited NMR restraints such as large proteins, oligonucleotides, or their complexes.     

High-resolution 3D structure determination of kaliotoxin by solid-state NMR spectroscopy

High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from (1)H/(1)H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 A and 1.3 A for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.     

Peristaltic transport of multilayered power-law fluids with distinct viscosities: a mathematical model for intestinal flows

This paper is concerned with the theoretical study of two-dimensional peristaltic flow of power-law fluids in three layers with different viscosities. The analysis is carried out under low Reynolds number and long wavelength approximations. The shapes of the interfaces are described by a system of non-linear algebraic equations which are solved numerically as streamlines. It is found that the non-uniformity in the intermediate and peripheral layers diminishes when the viscosity of the intermediate layer is increased and that of the outermost layer is kept unaltered for both the pseudo-plastic and dilatant fluids. Similar are the observations when the viscosity of the outermost layer is increased and that of the intermediate layer is kept fixed. The flow rate increases with the viscosities of the peripheral and the intermediate layers but the viscosity of the outermost layer is more effective. However, the knowledge of the effect of the viscosity of the intermediate layer facilitates us to achieve the required flow rate without disturbing the outermost layer. An increase in the flow behaviour index too favours larger flow rates. The trapping limits increase with the viscosity of the intermediate layer but decrease with the viscosity of the outermost layer and the flow behaviour index. Thus, a medicinal intervention that creates a more viscous intermediate layer and reduces pseudo plasticity may reduce constipation.