Forced folding and structural analysis of metastable proteins

A significant fraction of the proteins encoded by the human and other genomes appears to be significantly unfolded in vitro. This will undoubtedly hamper attempts to characterize their structure by classical crystallographic or solution NMR methods. Here we show that encapsulation of a metastable protein within the restricted volume a reverse micelle can be used to force fold the protein and allow its characterization by modern methods of NMR spectroscopy. This may have significant utility in the context of structural proteomics. In addition, variation of the inner volume of the reverse micelle can be used to probe the character of the manifold of unfolded states.     

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.     

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.     

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.     

Differential dynamical effects of macromolecular crowding on an intrinsically disordered protein and a globular protein: implications for in-cell NMR spectroscopy

In-cell NMR provides a valuable means to assess how macromolecules, with concentrations up to 300 g/L in the cytoplasm, affect the structure and dynamics of proteins at atomic resolution. Here an intrinsically disordered protein, alpha-synuclein (alphaSN), and a globular protein, chymotrypsin inhibitor 2 (CI2) were examined by using in-cell NMR. High-resolution in-cell spectra of alphaSN can be obtained, but CI2 leaks from the cell and the remaining intracellular CI2 is not detectable. Even after stabilizing the cells from leakage by using alginate encapsulation, no CI2 signal is detected. From in vitro studies we conclude that this difference in detectability is the result of the differential dynamical response of disordered and ordered proteins to the changes of motion caused by the increased viscosity in cells.     

Noncovalent encapsulation of cobaltocenium inside resorcinarene molecular capsules

The encapsulation of cobaltocenium (Cob+) inside hexameric molecular capsules of two different resorcinarenes was investigated in dichloromethane solution. Both 1H NMR spectroscopic and voltammetric experiments clearly reveal that Cob+ experiences encapsulation. Diffusion coefficient measurements obtained from PGSE NMR experiments indicate that the molecular capsules exist in dichloromethane solution in the absence of any cations. Bound and free Cob+ ions undergo slow exchange on the NMR time scale, but the bound Cob+ ions rotate and/or tumble freely inside the molecular capsules. Under experimental conditions suitable for voltammetry the encapsulation of Cob+ depends on the nature of the supporting electrolyte. Tetraalkylammonium hexafluorophosphate, tetrafluoroborate, and perchlorate supporting electrolytes prevent the encapsulation of Cob+, while tetraalkylammonium chloride and bromide salts allow it. The nature of the tetraalkylammonium cation plays a smaller role in the encapsulation. Finally, the structure of the resorcinarene also factors into the overall stability of the molecular assembly.     

Rotational Mechanism of Ammonium Ion in Water and Methanol?

Dynamics of ammonium and ammonia in solutions is closely related to the metabolism of ammoniac compounds, therefore plays an important role in various biological processes. NMR measurements indicated that the reorientation dynamics of NH₄⁺ is faster in its aqueous solution than in methanol, which deviates from the Stokes-Einstein-Debye rule since water has higher viscosity than methanol. To address this intriguing issue, we herein study the reorientation dynamics of ammonium ion in both solutions using numerical simulation and an extended cyclic Markov chain model. An evident decoupling between translation and rotation of methanol is observed in simulation, which results in the deviation of reorientation from the Stokes-Einstein-Debye rule. Slower hydrogen bond (HB) switchings of ammonium with methanol comparing to that with water, due to the steric effect of the methyl group, remarkably retards the jump rotation of ammonium. The observations herein provide useful insights into the dynamic behavior of ammonium in the heterogeneous environments including the protein surface or protein channels.     

Nematic reorientation in electric and magnetic fields

Homogeneous reorientation processes of two nematic liquid crystals in electric and magnetic fields have been observed using proton nuclear magnetic resonance spectroscopy (NMR). Using a recently developed experimental set-up, it is possible to study reorientation processes in liquid crystals by means of NMR experiments in a very flexible way. The time constant τ describing these processes has been determined as a function of the applied electric field. It emerges that the electric field cannot only be used to increase the reorientation time but also to slow the director reorientation by approximately one order of magnitude. Experimental data for 5CB and a fluorinated liquid crystal (BCH-5 FFF) are presented. The reorientation time measured as a function of the electric field can be used to calculate the rotational viscosity γ ₁. By repeating these experiments at different temperatures it was possible to investigate the temperature behaviour of γ ₁.     

Visualizing Proteins in Human Cells at Near-Physiological Concentrations with Sensitive 19F NMR Chemical Tags

In-cell NMR spectroscopy is an effective tool for observing proteins at atomic resolution in their native cellular environment. However, its utility is limited by its low sensitivity and the extensive line broadening caused by nonspecific interactions in the cells, which is even more pronounced in human cells due to the difficulty of overexpressing or delivering high concentrations of isotopically labeled proteins. Here, we present a high-sensitivity tag (wPSP-6F) containing two trifluoromethyl groups that can efficiently label globular proteins with molecular weights in the 6–40 kDa range under mild conditions. This tag allowed us to detect globular proteins in human cells at concentrations as low as 1.0 μM, which would not have been achievable with ¹⁵N or 3-fluorotyrosine labeling. Moreover, we detected conformational changes and interactions of proteins in the cellular environment. The new sensitive ¹⁹F NMR tag may significantly expand the scope of protein NMR in human cells.     

Opening a Can of Worm(‐like Micelle)s: The Effect of Temperature of Solutions of Functionalized Dipeptides

A simple heat/cool cycle can be used to significantly affect the properties of a solution of a low‐molecular‐weight gelator at high pH. The viscosity and extensional viscosity are increased markedly, leading to materials with very different properties than when the native solution is used.     

Protein-loaded PLGA–PEO blend nanoparticles: encapsulation, release and degradation characteristics

The aim of this work was to study the variables that affect the encapsulation and release of proteins from nanoparticles based on poly(lactic-co-glycolic acid; PLGA)–poloxamer and PLGA–poloxamine blend matrices, using bovine serum albumin (BSA) and immuno-γ-globulin (IgG) as model proteins. The nanoparticles were prepared by a solvent diffusion technique, and the studied variables were (1) PLGA molecular weight, (2) type of PEO-block copolymers, (3) protein loading, (4) pH and, (5) volume of the protein solution. Our results showed that the proteins can be efficiently incorporated into and released from the blend matrices. The type of the PEO derivative and the pH of the internal aqueous phase were the most important factors influencing protein encapsulation and release kinetics. Moreover, comparative degradation study of PLGA, PLGA–poloxamer and PLGA–poloxamine nanoparticles confirmed that the degradation and release characteristics of polyester particles can be improved by the incorporation of polyoxyethylene derivatives with different hydrophilia–lipophilia balance.     

Fast local backbone dynamics of encapsulated ubiquitin

Backbone dynamics of ubiquitin confined within AOT reverse micelles have been evaluated based on analysis of 15N NMR relaxation data. Results indicate that upon encapsulation the protein experiences a slight overall increase in the value of the order parameter, S2, indicating a restriction in the average amplitude of fast local N-H bond vector motion. The largest increases in S2 upon encapsulation were concentrated in the region of beta-sheet 2 and, additionally, at the transitions of secondary structure motifs and loop regions. In addition, statistical analysis of the residue average ratio of the 15N longitudinal and transverse NMR relaxation time constants indicates that chemical exchange contributions to relaxation are consistent with previous aqueous studies. Earlier studies have demonstrated that native protein structure can be maintained in the encapsulated state. These results presented here establish that the dynamical behavior of encapsulated ubiquitin is likewise nativelike and adds important new observations regarding the enhancement of protein stability under confinement.     

Conformational flexibility of a microcrystalline globular protein: order parameters by solid-state NMR spectroscopy

The majority of protein structures are determined in the crystalline state, yet few methods exist for the characterization of dynamics for crystalline biomolecules. Solid-state NMR can be used to probe detailed dynamic information in crystalline biomolecules. Recent advances in high-resolution solid-state NMR have enabled the site-specific assignment of (13)C and (15)N nuclei in proteins. With the use of multidimensional separated-local-field experiments, we report the backbone and side chain conformational dynamics of ubiquitin, a globular microcrystalline protein. The measurements of molecular conformational order parameters are based on heteronuclear dipolar couplings, and they are correlated to assigned chemical shifts, to obtain a global perspective on the sub-microsecond dynamics in microcrystalline ubiquitin. A total of 38 Calpha, 35 Cbeta and multiple side chain unique order parameters are collected, and they reveal the high mobility of ubiquitin in the microcrystalline state. In general the side chains show elevated motion in comparison with the backbone sites. The data are compared to solution NMR order parameter measurements on ubiquitin. The SSNMR measurements are sensitive to motions on a broader time scale (low microsecond and faster) than solution NMR measurements (low nanosecond and faster), and the SSNMR order parameters are generally lower than the corresponding solution values. Unlike solution NMR relaxation-based order parameters, order parameters for (13)C(1)H(2) spin systems are readily measured from the powder line shape data. These results illustrate the potential for detailed, extensive, and site-specific dynamic studies of biopolymers by solid-state NMR.     

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.     

新型聚磷酸酯药物控释材料的合成

采用溶液缩聚方法，制得含酪氨酸烷基酯的聚磷酸酯，再通过大分子反应分别得到主链带正、负电荷的聚磷酸酯，研究了这三类聚磷酸酯的体外降解行为，并对它们作为蛋白质的控制释放材料的性能进行了初步评价。     

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.     

New optically active poly(amide–imide)s from N-trimellitylimido-l-amino acid and 1,2-bis[4-aminophenoxy]ethane in the main chain: Synthesis and characterization

Six new optically active poly(amide–imide)s (PAIs) with good inherent viscosities were synthesized from the direct polycondensation reaction of N-trimellitylimido-l-amino acids with 1,2-bis[4-aminophenoxy]ethane by direct polycondensation in a medium consisting of N-methyl-2-pyrrolidone (NMP)/triphenyl phosphite (TPP)/calcium chloride (CaCl₂)/pyridine (py). Diamine was synthesized by using a two-step reaction. At first 1,2-bis[4-nitrophenoxy]ethane was prepared from the reaction of two equimolars 4-nitrophenol and one equimolar 1,2-dibromo ethane and the dinitro compound was reduced by using Pd/C. Also N-trimellitylimido-l-amino acids were synthesized by the condensation reaction of trimellitic anhydride with two equimolars of various l-amino acids in acetic acid solution. The polymerization reactions produced a series of optically active PAIs with a high yield and good inherent viscosity. The resulted polymers were fully characterized by means of FTIR and ¹H NMR spectroscopy, elemental analyses, inherent viscosity, specific rotation, solubility tests, thermogravimetric analysis (TGA), and a derivative of thermogravimetric (DTG) analysis.     

Characterization of globular protein solutions by dynamic light scattering, electrophoretic mobility, and viscosity measurements

In this work, physicochemical properties of two globular proteinsbovine serum albumin (BSA) having a molecular weight of 67 kDa and human serum albumin (HSA) having a molecular weight of 69 kDawere characterized. The bulk characteristics of these proteins involved the diffusion coefficient (hydrodynamic radius), electrophoretic mobility, and dynamic viscosity as a function of protein solution concentration for various pH values. The hydrodynamic radius data suggested an association of protein molecules, most probably forming compact dimers. Using the hydrodynamic diameter and the electropheretic mobility data allowed the determination of the number of uncompensated (electrokinetic) charges on protein surfaces. The electrophoretic mobility data were converted to zeta potential values, which allowed one to determine the isoelectric point (iep) of these proteins. It was found to be at pH 5.1 for both proteins, in accordance with previous experimental data and theoretical estimations derived from amino acid composition and p K values. To determine further the stability of protein solutions, dynamic viscosity measurements were carried out as a function of their bulk volume concentration for various pH values. The intrinsic viscosity derived from these measurements was interpreted in terms of the Brenner model, which is applicable to hard spheroidal particles. It was found that the experimental values of the intrinsic viscosity of these proteins were in good agreement with this model when assuming protein dimensions of 9.5 x 5 x 5 nm3 (prolate spheroid). The possibility of forming linear aggregates of association degree higher than 2 was excluded by these measurements. It was concluded that the combination of dynamic viscosity and dynamic light scattering can be exploited as a convenient tool for detecting not only the onset of protein aggregation in suspensions but also the form and composition of these aggregates.