Effective Hydrodynamic Radius Research Articles

Response of native and denatured hen lysozyme to high pressure studied by 15N/1H NMR spectroscopy

High-pressure (15)N/(1)H NMR techniques were used to characterize the conformational fluctuations of hen lysozyme, in its native state and when denatured in 8 M urea, over the pressure range 30--2000 bar. Most (1)H and (15)N signals of native lysozyme show reversible shifts to low field with increasing pressure, the average pressure shifts being 0.069 +/- 0.101 p.p.m. ((1)H) and 0.51 +/- 0.36 p.p.m. ((15)N). The shifts indicate that the hydrogen bonds formed to carbonyl groups or water molecules by the backbone amides are, on average, shortened by approximately 0.02 A as a result of pressure. In native lysozyme, six residues in the beta domain or at the alpha/beta domain interface have anomalously large nonlinear (15)N and (1)H chemical-shift changes. All these residues lie close to water-containing cavities, suggesting that there are conformational changes involving these cavities, or the water molecules within them, at high pressure. The pressure-induced (1)H and (15)N shifts for lysozyme denatured in 8 M urea are much more uniform than those for native lysozyme, with average backbone amide shifts of 0.081 +/- 0.029 p.p.m. ((1)H) and 0.57 +/- 0.14 p.p.m. ((15)N). The results show that overall there are no significant variations in the local conformational properties of denatured lysozyme with pressure, although larger shifts in the vicinity of a persistent hydrophobic cluster indicate that interactions in this part of the sequence may rearrange. NMR diffusion measurements demonstrate that the effective hydrodynamic radius of denatured lysozyme, and hence the global properties of the denatured ensemble, do not change detectably at high pressure.

Conformational changes of proteins in aqueous solution induced by temperature in the pre-melting region

In this paper we report dielectric measurements at radiofrequencies on aqueous solutions of three proteins (cytochrome, lysozyme and metmyoglobin) in the native state as a function of temperature in the interval 0–60°C. From the analysis of dielectric relaxation of the protein solutions, the effective hydrodynamic radius r and the electric dipole moment μ of the proteins were calculated. The results show that temperature causes continuous gradual changes of r and μ, with a maximum at a temperature that is different for each protein. This effect in the pre-melting region seems to be general and related to variations of thermodynamic parameters.

Effect of Trehalose on Alkaline Transition of Cytochrome-c

This work deals with a study of alkaline transition of ferricytochrome-c in water and in water−trehalose solutions using two different techniques: spectrophotometry in the region of the heme absorption spectrum and dielectric spectroscopy at radio frequencies. The first is a local probe and provides qualitative information on the identity of axial ligands to the heme iron. The second is able to reveal small variations in conformation and/or hydration state of the protein by two parameters: electric dipole moment and effective hydrodynamic radius. Optical absorption measurements of cytochrome-c in water as a function of pH show, in the basic range, a transition of the protein toward a partially denatured state, characterized by a displacement of methionine-S−Fe linkage in the heme. Dielectric measurements show that a simultaneous increase of the hydrodynamic radius and a decrease of the electric dipole moment of the protein accompany this alkaline transition. Thus, in water, conformational changes of cytochrome at basic pH result both in local structural changes in the heme pocket and in more global changes of the protein structure. In the water−trehalose solutions, optical measurements reveal a transition similar to that in water. On the contrary, dielectric parameters keep constant in the same pH range. Despite the displacement of the methionine-S−Fe linkage in the heme, the presence of trehalose stabilizes the global conformation of protein. This is coherent with the role of trehalose as bioprotector.

A compact monomeric intermediate identified by NMR in the denaturation of dimeric triose phosphate isomerase

The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spectroscopy in conjunction with optical spectroscopy. In the absence of denaturant, the hydrodynamic radius of 29.6(±0.25) Å and the substantial chemical shift dispersion evident in the NMR spectrum are consistent with the highly structured dimeric native state of the protein. On the addition of 2.2 M guanidine hydrochloride the effective hydrodynamic radius increases to 51.4(±0.43) Å, consistent with that anticipated for the polypeptide chain in a highly unstructured random coil state. In 1.1 M guanidine hydrochloride, however, the effective hydrodynamic radius is 24.0(±0.25) Å, a value substantially decreased relative to that of the native dimeric state but very close to that anticipated for a monomeric species with native-like compaction (23.5 Å). The lack of chemical shift dispersion indicates, however, that few tertiary interactions persist within this species. Far UV CD and intrinsic fluorescence measurements show that this compact intermediate retains significant secondary structure and that on average the fluorophores are partially excluded from solvent. Such a species could be important in the formation of dimeric TIM from its unfolded state.

The hydrodynamic characterization of waxy maize amylopectin in 90% dimethyl sulfoxide–water by analytical ultracentrifugation, dynamic, and static light scattering

Static light scattering of high amylopectin waxy maize starch gently dispersed in 90% dimethyl sulfoxide–water yielded a weight average molecular weight M w and radius of gyration R g of 560×10 6 g/mol and 342 nm, respectively. To obtain an independent hydrodynamic characterization of these solutions, we measured the sedimentation coefficient for the main component in an analytical ultracentrifuge. The value of s 0, the infinite dilution sedimentation coefficient, was 199 S. The translational diffusion coefficient D 0 in very dilute solutions was measured by dynamic light scattering at 90° and found to be 2.33×10 −9 cm 2/s. An effective hydrodynamic radius R h was calculated from this diffusion constant using the Stokes–Einstein equation and found to be 348 nm. The structure-related parameter ρ= R g/ R h was calculated to be 0.98. The weight average molecular weight calculated from the Svedberg equation using the values measured for s 0 and D 0 was 593×10 6 g/mol. This result is in reasonable agreement with the light scattering results. As light scattering results are subject to experimental errors due to the possibility of dust contamination, the presence of microgel or aggregates, and the questionable applicability of light scattering theory to interpret results for macromolecular sizes approaching the wave length of light used as a source for scattering, it is advisable to have corroborating hydrodynamic data when possible to further validate light scattering results in this very high molecular weight range.

Schizosaccharomyces pombe Aps1, a diadenosine 5',5' "-P1, P6-hexaphosphate hydrolase that is a member of the nudix (MutT) family of hydrolases: cloning of the gene and characterization of the purified enzyme.

The fission yeast Schizosaccharomyces pombe contains a gene on chromosome I that encodes a hypothetical nudix hydrolase, YA9E. The gene, designated aps1, has been cloned and the protein has been purified from Escherichia coli with a yield of 10 mg of Aps1/L of culture. Aps1, composed of 210 amino acids with a calculated molecular mass of 23 724 Da, behaves as a monomer with a sedimentation coefficient of 1.92 S as determined by analytical ultracentrifugation. The effective hydrodynamic radius is about 29 A as determined by both analytical ultracentrifugation and gel-filtration chromatography. Aps1, whose expression was detected in S. pombe by Western blotting, is an enzyme that catalyzes the hydrolysis of dinucleoside oligophosphates, with Ap6A and Ap5A being the preferred substrates. The major reaction products are ADP and p4A from Ap6A and ADP and ATP from Ap5A. Values of Km for Ap6A and Ap5A are 19 microM and 22 microM, respectively, and the corresponding values of kcat are 2.0 s-1 and 1.7 s-1, respectively. The enzyme has limited activity on Ap4A and negligible activity on Ap3A, ADP-ribose, and NADH. Aps1 catalyzes the hydrolysis of mononucleotides with decreasing activity in order from p5A to AMP. Optimal activity with Ap6A as substrate is observed at pH 7.6 and in the presence of 0.1-1 mM MnCl2. Aps1 is the first nudix hydrolase isolated from S. pombe, and it is the first enzyme identified with this specific substrate specificity and reaction products.

Computer simulation and mode coupling theory study of the effects of specific solute–solvent interactions on diffusion: Crossover from a sub-slip to a super-stick limit of diffusion

In many experimental situations, the interaction potential between the tagged solute and the solvent molecules is often different from that between the two solvent molecules. In such cases, the Stokes–Einstein relation attempts to describe the self-diffusion of the solute in terms of an effective hydrodynamic radius which, along with the hydrodynamic boundary condition (slip or stick), are varied to fit the experimental results. Extensive molecular dynamics (MD) simulations have been carried out to obtain the diffusion coefficient by varying interaction between the solute and the solvent. It is found that when this interaction is more repulsive than that between solvent–solvent, the diffusion can be significantly faster, leading to a complete breakdown of the Stokes–Einstein relation. In the limit of strong attractive interaction, we recover a dynamic version of the solvent–berg picture. The diffusion coefficient of the solute is found to depend strongly and nonlinearly on the magnitude of this specific interaction. The velocity correlation function also shows an interesting dependence on the sign and magnitude of the specific interaction. Another potentially important observation is that the specific solute–solvent interaction can induce a crossover from a sliplike to a stick-like diffusion, if one still uses the hydrodynamic language. Mode coupling theory analysis of the friction shows that the change in it originates largely from the modification of the binary component of the total friction. This is because the cage structure around the solute is modified due to the specific solute–solvent interaction, which directly affects the binary dynamics.

Temperature-induced conformational changes of native lysozyme in aqueous solution studied by dielectric spectroscopy

We report dielectric measurements at radiofrequencies on lysozyme in aqueous solution at two values of pH (3.5 and 6) as a function of temperature in the interval 5–55°C. From the analysis of the dielectric relaxation of the protein solution, the effective hydrodynamic radius r and the electric dipole moment μ of the protein were calculated. The results show that temperature causes continuous gradual changes of r and μ with a maximum at 25–30°C where the Gibbs free energy for native lysozyme shows an analogous trend. We suggest that the gradual variations of r and μ are the manifestation of a redistribution of microscopic state populations of the protein within the same macroscopic native state.

Characterization of the solution properties of Pichia farinosa killer toxin using PGSE NMR diffusion measurements.

The solution behaviour with respect to pH and NaCl concentration of the tertiary structure and propensity for aggregation of salt- mediated killer toxin (SMKT) from Pichia farinosa was examined using pulsed-gradient spin-echo NMR diffusion measurements. It was found that in 0.15m NaCl the tertiary structure of SMKT was constant below pH 5.0, with the native SMKT existing as an unaggregated heterodimer containing the beta-subunit in a compactly folded form. However, above pH 5.0 the beta-subunit dissociated and lost its compact structure, becoming a random coil with an approximately 37% increase in effective hydrodynamic radius. To determine the effects of NaCl concentration on the tertiary structure of SMKT, diffusion measurements were performed at pH 3.5 and NaCl concentrations up to 2M. Both the tertiary structure and aggregation state of SMKT were found to be insensitive to the salt concentration which indicates that the activity of the toxin is not a direct result of salt-protein interactions.

Modeling and Prediction of Electrophoretic Mobilities in Capillary Electrophoresis: Separation of Alkylpyridines

The relative electrophoretic mobilities of a series of closely related alkylpyridines in capillary electrophoresis have been predicted by proposing that they experience a preferred orientation under the influence of the applied electrical field. This means that analytes with the same van der Waals volumes can exhibit different effective hydrodynamic radii to motion through the buffer solution. Additional terms for these differences in apparent volume and for the forces acting to orient the analytes can be calculated from the molecular structures and influence the dominant effect of the total volume. The model could correctly predict the relative mobility of structural, positional, and geometric isomers of alkylated and unsaturated pyridines.

Influence of pH on lysozyme conformation revealed by dielectric spectroscopy

In this paper we report permittivity measurements in the frequency range 10 5–10 8 Hz, at the fixed temperature of 20°C, on lysozyme dissolved in water and in a water–ethanol mixture (ethanol concentration 0.1 molar fraction) varying the pH of the solutions from acid (≅pH 2) to basic (≅pH 10). The experimental data were fitted with the Cole–Cole equation and from the dispersion parameters the effective hydrodynamic radius and the electric dipole moment of the protein in different conditions of pH were calculated. The results confirm a conformational effect induced on lysozyme by ethanol, reported in our previous work. For pH in the range 4–6, where the enzymatic activity of lysozyme reaches its maximum, our results indicate a stable conformation of the protein. Out of this pH interval aggregation and expansion processes are present.

Amyloid fibril formation by an SH3 domain.

The SH3 domain is a well characterized small protein module with a simple fold found in many proteins. At acid pH, the SH3 domain (PI3-SH3) of the p85alpha subunit of bovine phosphatidylinositol 3-kinase slowly forms a gel that consists of typical amyloid fibrils as assessed by electron microscopy, a Congo red binding assay, and x-ray fiber diffraction. The soluble form of PI3-SH3 at acid pH (the A state by a variety of techniques) from which fibrils are generated has been characterized. Circular dichroism in the far- and near-UV regions and 1H NMR indicate that the A state is substantially unfolded relative to the native protein at neutral pH. NMR diffusion measurements indicate, however, that the effective hydrodynamic radius of the A state is only 23% higher than that of the native protein and is 20% lower than that of the protein denatured in 3.5 M guanidinium chloride. In addition, the A state binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid, which suggests that SH3 in this state has a partially formed hydrophobic core. These results indicate that the A state is partially folded and support the hypothesis that partially folded states formed in solution are precursors of amyloid deposition. Moreover, that this domain aggregates into amyloid fibrils suggests that the potential for amyloid deposition may be a common property of proteins, and not only of a few proteins associated with disease.

Salt-Induced Sphere-to-Disk Transition of Octadecyltrimethylammonium Bromide Micelles

We have used surface tension measurements, differential scanning calorimetry (DSC), dynamic light scattering (DLS), and cryo-transmission electron microscopy (cryo-TEM) to investigate the dynamic and structural behavior of octadecyltrimethylammonium bromide (C18TAB) micelles in water and NaBr solution. The surface tension data for fixed C18TAB concentrations of 25 mM and varied NaBr additions (0−50 mM) shows that the critical micelle concentration (cmc) increases after an initial decrease at 0.5 mM NaBr. This unusual effect has been explained using results from DSC and DLS. At low salt concentrations (below ca. 25 mM) the relaxation time distribution is bimodal with a dominant fast mode due to spherical micelles. Above ca. 35 mM NaBr disklike structures are favored and the relaxation time distribution is more closely unimodal. The postulated sphere-to-disk transition is supported by cryo-TEM micrographs. A pronounced increase in the micellar effective hydrodynamic radius (RH) is observed as the NaBr conce...

Conformational changes of lysozyme in water-ethanol mixtures

In this paper, we report dielectric spectroscopy and optical measurements on lysozyme in water-ethanol mixtures as a function of alcohol concentration in the water-rich region of composition (cosolvent mole fraction X⩽0.25). Dielectric measurements show a well defined relaxation curve in the MHz region, from which we obtain the electric dipole moment μ and the effective hydrodynamic radius r of the protein. UV melting measurements with estimation of the van't Hoff enthalpy are also reported. Molar ellipticity measurements were performed to test the α-helix content. The results indicate that alcohol promotes a more tightly folded conformation of the protein. Our measurements show a correlation between the effects of ethanol on the conformational properties of lysozyme and changes in the properties of solvent, as inferred from previous adiabatic compressibility and IR data on water-ethanol mixtures.

Effect of Regular Anisotropic Permanent Bending on the Diffusional Spinning and Fluorescence Polarization Anisotropy of Short DNA Fragments Studied by Brownian Dynamics Simulation

The effects of regular permanent bends on the rotational Brownian motions of flexurally and torsionally deformable DNA models are investigated by Brownian dynamics simulations. Directionality of the permanent bends is maintained by anisotropic bending terms in the intersubunit potential. The effects of such bends on the effective hydrodynamic radius for diffusional spinning and on the fluorescence polarization anisotropy (FPA) are determined by comparing simulated results for three different model potentials: (1) a uniform isotropic bending potential with a straight resting geometry (I-model); (2) a uniform isotropic plus anisotropic permanent bending potential, which has the same average projection of a bond vector onto its predecessor as the I-model and which exhibits regular permanent bends (A-model); and (3) a mostly isotropic model, but with a single 90° anisotropic permanent bend at its center (L-model). A 72 base pairs (bp) I-model exhibits practically the same hydrodynamic radius for azimuthal spinning as the corresponding straight cylinder, whereas the A-model exhibits a slightly (3%) larger value over a range of filament lengths. The large directional permanent bend of the L-model substantially increases the hydrodynamic radius for diffusional spinning. However, when the anisotropic bending potential is omitted from either the A- or L-models, the permanent bends become nondirectional and have no significant effect on diffusional spinning, which evidently takes place via a uniform speedometer cable rotation. The FPA of the A-model indicates significantly less depolarization than is obtained for the I-model at any given time. The A-model FPA curves can be satisfactorily reproduced by suitably parametrizing the I-model so that (1) the effective hydrodynamic radius for azimuthal rotation is increased to emulate the effect of permanent bends; (2) the dynamic bending rigidity is chosen to emulate the short-time bending amplitude of the A-model; and (3) the rotational diffusion coefficient for end-over-end tumbling is chosen to reflect the total persistence length, including the effect of permanent bends.

Effect of Sodium Dodecyl Sulfate on Coil-Globule Transitions of Poly(N-isopropyl acrylamide)

Mechanical properties of aqueous solutions of poly(N-isopropylacrylamide) (PNIPAM) were studied over a temperature range from 25° C to 40° C using a Zimm viscometer. For a PNIPAM/water system a remarkable increase in specific viscosity, η sp, was found at about 31° C. But in the case of PNIPAM (0.1 g/d l )/sodium dodecyl sulfate (SDS) (0.03 g/d l )/water system a clear decrease in η sp from 0.27 to 0.12 was observed on going through that temperature. The translational diffusion constant was also measured by dynamic light scattering (DLS), and a decrease in the effective hydrodynamic radius by a factor of 1/3 was observed over the same temperature range. These results suggest that SDS prevents the aggregation of polymers without hindering the intramolecular phase transitions.

Characterization of branched polyethyleneimine by laser light scattering and viscometry

A series of seven fractions of branched polyethyleneimine (BPEI) spanning a molecular weight range 1.7 × 10 4−5.3 × 10 6 g mol −1 were characterized by means of laser light scattering and viscometry at 35°C. The weight-average molecular weight, M W, the second virial coefficient, A 2 ∗ , the z-average radius of gyration, R G, the z-average effective hydrodynamic radius, R H, the intrinsic viscosity, [η], and the Huggins coefficient, K H, were determined in 1 M NaCl aqueous mixture solvent. Based upon these experimental results, the scaling relations between M W and the various kinds of radii were established. The exponents of the relations appeared to be much smaller than the generally accepted value for a linear polymer chain (for example, the Mark—Houwink equation; [ η] = 0.513 M W 0.31). Also some ratios such as R G/ R H and R G/ R T showed a molecular weight dependence, where R T [= (3A 2 ∗M W 2/16πN A ) 1 3 ] was defined as the thermodynamic radius. Although quantitative comparison with theories was somewhat restricted due to the slightly high index of polydispersity ( M W/ M n = 1.8−2.2) of the BPEI fractions, it was found that the structure of BPEI seemed to be randomly branched and became more compact with increasing molecular weight.

A dynamic measure of order in structural glasses

Although some patterns of physical behavior are common in the glass transition and in the properties of supercooled liquids and glasses (characteristic viscoelasticity, temperature dependence of viscosity and relaxation times, property evolution through “physical aging”, difficulties in performing equilibrium measurements or simulations, etc.), it is difficult to arrive at a definition of the glass transition which distinguishes it from other phenomena exhibiting similar features. The present paper addresses this problem by defining a dynamical measure of order involving the average “shape” of particle trajectories in supercooled liquids. This dynamic order parameter should provide a measure of “closeness” to the glass transition and some indirect insights into the physical nature of supercooled liquids and glasses. Arguments are given that the proposed dynamic measure of order [“generalized capacity”, C( T)] is related to the temperature-dependent “effective hydrodynamic radius” R H ( T) measured in supercooled liquids and model numerical calculations are given to support this view. Some consequences of the intermittent particle motion at low temperatures for stress relaxation are also discussed.

Solution behavior of buckminsterfullerene (C60) in benzene

Solution characterization of buckminsterfullerene C60 in benzene was carried out by laser light scattering. Static and dynamic light scattering (SLS and DLS) experiments were accomplished by using an incident laser beam of 790 nm wavelength, and a fiber optic probe which was coupled to a RCA C31034 photomultiplier tube sensitive to the near ir wavelength. The effective hydrodynamic radius of C60 in benzene solution determined by using DLS was 6.4±0.1 Å, in close agreement with the distance measured from the center of the solvated benzene to the center of the benzene-solvated C60 (C60⋅4C6H6):6.7∼6.8 Å as evaluated from the single crystal structure by x-ray diffraction. C60 was found to aggregate slowly even in fairly dilute solution concentrations ranging from 0.78 to 1.39 mg/mL at temperatures 23∼27 °C. Based on the high specific refractive index increment of C60 in benzene, the molar mass of the C60 aggregates could be estimated. Together with the radius of gyration, the hydrodynamic radius, and its size distribution, the kinetics of the aggregation process was investigated. The aggregation of C60 in benzene solution was reversible, i.e., the C60 aggregates could be dispersed simply by hand shaking the solution. The light-scattering intensity results indicated that the structure of C60 aggregates could be described as a fractal with a dimension of 2.10 and the aggregation kinetics exhibited essentially an exponential behavior.

Solvent Stokes-Einstein violation in aqueous protein solutions.

The Stokes-Einstein equation is applied to the water self-diffusion coefficient D in human serum albumin protein solutions. A linear trend for D as a function of T/\ensuremath{\eta} is found for all the protein concentrations investigated. However, the indication of a violation of the Stokes-Einstein equation is found in the protein concentration dependence of the effective hydrodynamic radius of water. The deviation of the experimental NMR water self-diffusion and viscosity data from the hydrodynamic Stokes-Einstein relation is found to be consistent with an enhancement of the solvent structure in the vicinity of the protein surface.