Ion-specificity and surface water dynamics in protein solutions.
ABSTRACT: Ion-specific effects at the protein surface are investigated here in light of the changes they infer to surface water dynamics, as observed by 1H NMR relaxation (at 20 MHz). Two well-known proteins, hen egg-white lysozyme (LZM) and bovine serum albumin (BSA), show qualitatively opposite trends in the transverse relaxation rate, R2(1H), along a series of different monovalent salt anions in the solution. Presence of salt ions increases R2(1H) in the case of lysozyme and diminishes it in the case of BSA. The effect magnifies for larger and more polarizable ions. The same contrasting effect between the two proteins is observed for protein-solvent proton exchange. This hints at subtle effects ion-binding might have on the accessibility of water surface sites on the protein. We suggest that the combination of the density of surface charge residues and surface roughness, at the atomic scale, dictates the response to the presence of salt ions and is proper to each protein. Further, a dramatic increase in R2(1H) is found to correlate closely with the formation of protein aggregates. The same ordering of salts in their ability to aggregate lysozyme, as seen previously by cloud point measurements, is reproduced here by R2(1H). 1H NMR relaxation data is supplemented by 35Cl and 14N NMR relaxation for selected salt ions to probe the ion-binding itself.
Project description:New colorimetric receptors R1 and R2 with varied positional substitution of a cyano and nitro signaling unit having a hydroxy functionality as the hydrogen bond donor site have been designed, synthesized and characterized by FTIR, 1H NMR spectroscopy and mass spectrometry. The receptors R1 and R2 exhibit prominent visual response for F- and AcO- ions allowing the real time analysis of these ions in aqueous media. The formation of the receptor-anion complexes has been supported by UV-vis titration studies and confirmed through binding constant calculations. The anion binding process follows a first order rate equation and the calculated rate constants reveal a higher order of reactivity for AcO- ions. The 1H NMR titration and TDDFT studies provide full support of the binding mechanism. The Hg2+ and F- ion sensing property of receptor R1 has been utilized to arrive at "AND" and "INHIBIT" molecular logic gate applications.
Project description:1H Nuclear magnetic resonance (NMR) relaxometry was exploited to investigate the dynamics of solid proteins. The relaxation experiments were performed at 37 °C over a broad frequency range, from approximately 10 kHz to 40 MHz. Two relaxation contributions to the overall 1H spin-lattice relaxation were revealed; they were associated with 1H-1H and 1H-14N magnetic dipole-dipole interactions, respectively. The 1H-1H relaxation contribution was interpreted in terms of three dynamical processes occurring on timescales of 10-6 s, 10-7 s, and 10-8 s, respectively. The 1H-14N relaxation contribution shows quadrupole relaxation enhancement effects. A thorough analysis of the data was performed revealing similarities in the protein dynamics, despite their different structures. Among several parameters characterizing the protein dynamics and structure (e.g., electric field gradient tensor at the position of 14N nuclei), the orientation of the 1H-14N dipole-dipole axis, with respect to the principal axis system of the electric field gradient, was determined, showing that, for lysozyme, it was considerably different than for the other proteins. Moreover, the validity range of a closed form expression describing the 1H-14N relaxation contribution was determined by a comparison with a general approach based on the stochastic Liouville equation.
Project description:The main particularities of sulfonate groups hydration, water molecule, and alkaline metal cation translation mobility were revealed by nuclear magnetic resonance (NMR) and ionic conductivity measurements techniques in cation-exchange membranes MSC based on cross-linked sulfonated polystyrene (PS) grafted on polyethylene with ion-exchange capacity of 2.5 mg-eq/g. Alkaline metal cation hydration numbers (h) calculated from temperature dependences of 1H chemical shift of water molecule for membranes equilibrated with water vapor at RH = 95% are 5, 6, and 4 for Li+, Na+, and Cs+ ions, respectively. These values are close to h for equimolar aqueous salt solutions. Water molecules and counter ions Li+, Na+, and Cs+ diffusion coefficients were measured by pulsed field gradient NMR on the 1H, 7Li, 23Na, and 133Cs nuclei. For membranes as well as for aqueous chloride solutions, cation diffusion coefficients increased in the following sequence: Li+ < Na+ < Cs+. Cation and water molecule diffusion activation energies in temperature range from 20 °C to 80 °C were close to each other (about 20 kJ/mol). The cation conductivity of MSC membranes is in the same sequence, Li+ < Na+ < Cs+ << H+. The conductivity values calculated from the NMR diffusion coefficients with the use of the Nernst-Einstein equation are essentially higher than experimentally determined coefficients. The reason for this discrepancy is the heterogeneity of membrane pore and channel system. Ionic conductivity is limited by cation transfer in narrow channels, whereas the diffusion coefficient characterizes ion mobility in wide pores first of all.
Project description:In this work, boundary element methods are used to model the electrophoretic mobility of lysozyme over the pH range 2-6. The model treats the protein as a rigid body of arbitrary shape and charge distribution derived from the crystal structure. Extending earlier studies, the present work treats the equilibrium electrostatic potential at the level of the full Poisson-Boltzmann (PB) equation and accounts for ion relaxation. This is achieved by solving simultaneously the Poisson, ion transport, and Navier-Stokes equations by an iterative boundary element procedure. Treating the equilibrium electrostatics at the level of the full rather than the linear PB equation, but leaving relaxation out, does improve agreement between experimental and simulated mobilities, including ion relaxation improves it even more. The effects of nonlinear electrostatics and ion relaxation are greatest at low pH, where the net charge on lysozyme is greatest. In the absence of relaxation, a linear dependence of mobility and average polyion surface potential, (lambda zero)s, is observed, and the mobility is well described by the equation [formula: see text] where epsilon 0 is the dielectric constant of the solvent, and eta is the solvent viscosity. This breaks down, however, when ion relaxation is included and the mobility is less than predicted by the above equation. Whether or not ion relaxation is included, the mobility is found to be fairly insensitive to the charge distribution within the lysozyme model or the internal dielectric constant.
Project description:A high resolution NMR structure of hen lysozyme has been determined using 209 residual 1H-15N dipolar coupling restraints from measurements made in two different dilute liquid crystalline phases (bicelles) in conjunction with a data set of 1632 NOE distance restraints, 110 torsion angle restraints, and 60 hydrogen bond restraints. The ensemble of 50 low-energy calculated structures has an average backbone RMSD of 0.50+/-0.13A to the mean structure and of 1.49+/-0.10A to the crystal structure of hen lysozyme. To assess the importance of the dipolar coupling data in the structure determination, the final structures are compared with an ensemble calculated using an identical protocol but excluding the dipolar coupling restraints. The comparison shows that structures calculated with the dipolar coupling data are more similar to the crystal structure than those calculated without, and have better stereochemical quality. The structures also show improved quality factors when compared with additional dipolar coupling data that were not included in the structure calculations, with orientation-dependent 15N chemical shift changes measured in the bicelle solutions, and with T1/T2 values obtained from 15N relaxation measurements. Analysis of the ensemble of NMR structures and comparisons with crystal structures, 15N relaxation data, and molecular dynamics simulations of hen lysozyme provides a detailed description of the solution structure of this protein and insights into its dynamical behavior.
Project description:Inter-protein interactions in solution affect the auto-correlation function of Brownian tumbling not only in terms of a simple increase of the correlation time, they also lead to the appearance of a weak slow component ("long tail") of the correlation function due to a slowly changing local anisotropy of the microenvironment. The conventional protocol of correlation time estimation from the relaxation rate ratio R1/R2 assumes a single-component tumbling correlation function, and thus can provide incorrect results as soon as the "long tail" is of relevance. This effect, however, has been underestimated in many instances. In this work we present a detailed systematic study of the tumbling correlation function of two proteins, lysozyme and bovine serum albumin, at different concentrations and temperatures using proton field-cycling relaxometry combined with R1? and R2 measurements. Unlike high-field NMR relaxation methods, these techniques enable a detailed study of dynamics on a time scale longer than the normal protein tumbling correlation time and, thus, a reliable estimate of the parameters of the "long tail". In this work we analyze the concentration dependence of the intensity and correlation time of the slow component and perform simulations of high-field (15)N NMR relaxation data demonstrating the importance of taking the "long tail" in the analysis into account.
Project description:PURPOSE:Dynamic in-situ proton (1H) magnetic resonance imaging (MRI) and 1H T2-relaxometry experiments are described in an attempt to: (i) understand the physical processes, that occur during the reconstitution of lyophilized bovine serum albumin (BSA) and monoclonal antibody (mAb) proteins; and (ii) objectify the reconstitution time. METHODS:Rapid two-dimensional 1H MRI and diffusion weighted MRI were used to study the temporal changes in solids dissolution and characterise water mass transport characteristics. One-shot T2 relaxation time measurements were also acquired in an attempt to quantify the reconstitution time. Both MRI data and T2-relaxation data were compared to standard visual observations currently adopted by industry. The 1H images were further referenced to MRI calibration data to give quantitative values of protein concentration and, percentage of remaining undissolved solids. RESULTS:An algorithmic analysis of the 1H T2-relaxation data shows it is possible to classify the reconstitution event into three regimes (undissolved, transitional and dissolved). Moreover, a combined analysis of the 2D 1H MRI and 1H T2-relaxation data gives a unique time point that characterises the onset of a reconstituted protein solution within well-defined error bars. These values compared favourably with those from visual observations. Diffusion weighted MRI showed that low concentration BSA and mAb samples showed distinct liquid-liquid phase separation attributed to two liquid layers with significant density differences. CONCLUSIONS:T2 relaxation time distributions (whose interpretation is validated from the 2D 1H MR images) provides a quick and effective framework to build objective, quantitative descriptors of the reconstitution process that facilitate the interpretation of subjective visual observations currently adopted as the standard practice industry.
Project description:The use of a squaramide-based ion pair receptor offers a solution to the very challenging problem of extraction and transport of extremely hydrated sulfate salt. Herein we demonstrate for the first time that a neutral receptor is able not only to selectively extract but also to transport sulfates in the form of an alkali metal salt across membranes and to do so in a cooperative manner while overcoming the Hofmeister bias. This was made possible by an enhancement in anion binding promoted by cation assistance and by diversifying the stoichiometry of receptor complexes with sulfates and other ions. The existence of a peculiar 4:1 complex of receptor 2 with sulfates in solution was confirmed by UV-vis and 1H NMR titration experiments, DOSY and DLS measurements, and supported by solid-state X-ray measurements. By varying the separation technique and experimental conditions, it was possible to switch the depletion of the aqueous layer into extremely hydrophilic or less lipophilic salts, thus obtaining the desired selectivity.
Project description:Molecular dynamics was used to investigate ion surface propensities in NaCl, KBr, and CsI solutions with an MP2-based force field. Although NaCl is found to be strongly repelled from the liquid-vapor interface, softer ions, such as I-, penetrate closely to the interface. Despite the surface penetration, the concentration of CsI near the interface is still lower than that in the bulk, thus leading to no surface enrichment. The salt concentration is found to affect relative surface propensities of the ions. More significant surface penetration is observed at higher salt concentrations. Softer ions at higher concentrations form a complex multilayer arrangement that can not be characterized as a simple surface bilayer. The simulated ion distributions explain the spectroscopic evidence of surface perturbation by soft ions with a negative surface excess consistent with an increased surface tension of salt solutions.
Project description:BACKGROUND: Hydrophobic biopolymers such as polylactide-co-glycolide (PLGA, 85:15) have been extensively explored as scaffolding materials for tissue engineering applications. More recently, electrospun microfiber-based and nanofiber-based scaffolds of PLGA have received increased attention because they act as physical mimics of the fibrillar extracellular matrix. However, the hydrophobicity of the PLGA microfiber surface can limit its use in biomedical applications. Therefore, in a previous study, we fabricated Pluronic(®) F-108 (PF-108)-blended PLGA microfibrous scaffolds that alleviated the hydrophobicity associated with PLGA by enriching the surface of microfibers with the ethylene oxide units present in PF-108. METHODS: In this study, we report the influence of the extent of surface enrichment of PLGA microfibers on their interaction with two model proteins, ie, bovine serum albumin (BSA) and lysozyme. BSA and lysozyme were adsorbed onto PLGA microfiber meshes (unmodified and modified) and studied for the amount, secondary structure conformation, and bioactivity of released protein. RESULTS: Irrespective of the type of protein, PF-108-blended PLGA microfibers showed significantly greater protein adsorption and release than the unblended PLGA samples. However, in comparison with BSA, lysozyme showed a 7-9-fold increase in release. The Fourier transform infrared spectroscopy studies for secondary structure determination demonstrated that irrespective of type of microfiber surface (unblended or blended), adsorbed BSA and lysozyme did not show any significant change in secondary structure (?-helical content) as compared with BSA and/or lysozyme in the free powder state. Further, the bioactivity assay of lysozyme released from blended PLGA microfiber meshes demonstrated 80%-85% bioactivity, indicating that the process of adsorption did not significantly affect biological activity. Therefore, this study demonstrated that the decreased hydrophobicity of blended PLGA microfibrous meshes not only improved the amount of protein adsorbed (lysozyme and BSA) but also maintained the secondary structure and bioactivity of the adsorbed proteins. CONCLUSION: Modulating the hydrophobicity of PLGA via blending with PF-108 could be a viable strategy to improve its interaction with proteins and subsequent cell interaction in tissue engineering applications.