Structure and dynamics of M13mp19 circular single-strand DNA: effects of ionic strength

Dynamic and static light scattering, CD, and optical melting experiments have been conducted on M13mp19 viral circular single-strand DNA as a function of NaCl concentration. Over the 10,000-fold range in concentration from 100 microM to 1.0 M NaCl, the melting curves and CD spectra indicate an increase in base stacking and stability of stacked regions with increased salt concentration. Analysis of dynamic light scattering measurements of the single-strand DNA solutions as a function of K2 from 1.56 to 20 X 10(10) cm-2 indicates the collected autocorrelation functions are biexponential, thus revealing the presence of two decaying dynamic components. These components are taken to correspond to (1) global translational motions of the molecular center of mass and (2) motions of the internal molecular subunits. From the evaluated relaxation rates of these components, diffusion coefficients D0 and Dplat are determined. The center of mass translational diffusion coefficient D0, varies in a nonmonotonic manner, by 10%, from 3.75 X 10(-8) to 3.39 X 10(-8) cm2/s over the NaCl concentration range from 100 microM to 1.0 M. Likewise, the radius of gyration RG, obtained from static light scattering experiments, varies by 15% from 699 to 830 A over the same NaCl range Dplat, the diffusion coefficient of the internal subunits, displays a different dependence on the NaCl concentration and decreases, by nearly 22% in a titratable fashion, from 12.46 X 10(-8) to 10.26 X 10(-8) cm2/s, when the salt is increased from 100 microM to 1.0 M. A semiquantitative interpretation of these results is provided by analysis of the light scattering data in terms of the circular Rouse-Zimm chain. Rouse-Zimm model parameters are estimated from the experimental results, assuming the circular chains are composed of a fixed number of Gaussian segments, N + 1 = 15. The rms displacement of the internal segments, b, is estimated to be the smallest (442 A) in 100 mM NaCl. Increases of b to 467 A in 100 microM and 524 A in 1.0 M NaCl are observed. Meanwhile, the hypothetical friction factor of the internal subunits, f, progressively increases as the NaCl concentration is raised. It is inferred from the evaluated Rouse-Zimm model parameters that both the static flexibility of the circular chain and diffusive displacements of the internal subunits decrease with increases in NaCl concentration from 100 mM to 1.0 M.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of molecular weight determination of sodium alginate by sedimentation-diffusion and light scattering

The weight average molecular weight, M̄_w, of sodium alginates were determined by the sedimentation-diffusion technique using photon correlation spectroscopy rather than boundary spreading in the analytical ultracentrifuge to determine the translational diffusion coefficients. This enables the diffusion coefficients to be determined easily and accurately. Excellent correlation is found between the observed zero concentration translational diffusion coefficient, D_O and the M̄_W values in a emphirical power law. The M̄_W obtained by sedimentation-diffusion and laser light scattering compare very favourably. The concentration dependence of the photon correlation spectroscopy data allowed determination of the coil overlap concentration, c*. The inverse proportionality of c* to both M̄_W and [η] is demonstrated.     

Dynamic Light Scattering Application to Study Protein Interactions in Electrolyte Solutions

The concentration dependence of the diffusion coefficient of particles suspended in solution depends primarily on the occupied volume fraction and on repulsive and attractive forces. This dependency is expressed by the interaction parameter, which can be assessed experimentally by light scattering measurements and have been determined for the diffusion coefficient of BSA under different salt concentration conditions in the present work. The result shows that the diffusion coefficient of protein grows up with increasing protein concentration, and when the ionic strength turns up gradually the diffusion coefficient decreases with protein concentrations increasing. The concentration dependence of BSA diffusion coefficients is interpreted in the context of a two-body potential of mean force, which includes repulsive hard-sphere and Coulombic interactions and attractive dispersion. With the increase of ionic strength, Debye screening decreases, protein interaction changes from repulsion to attraction, and protein begins to aggregate. By means of the concentration dependence of BSA diffusion coefficients, one can obtain the parameters of protein interactions and can find that protein bears a net effective charge of –9.0 e and has a Hamaker constant of 2.8k_BT. This work demonstrates that DLS is an effective technique of studying protein interactions.     

Salt dependent changes in structure and dynamics of circular single stranded DNA of filamentous phages of Escherichia coli

We have analyzed the static and dynamic behaviour of the circular single stranded DNA of the filamentous Escherichia coli phages F1 and M13mp8 in solution as a function of salt concentration using static and dynamic light scattering and sedimentation analysis in the analytical ultracentrifuge. We show by static light scattering that native and denatured single stranded DNA behave like a randomly coiled macromolecule at all salt concentrations used. The size of the native single stranded DNA is governed by the formation of secondary structures. While the radius of gyration decreases with increasing salt concentration the translational diffusion of the center-of-mass of native single stranded DNA and the sedimentation coefficient increase with increasing salt concentration in a biphasic manner. Below 100 mM monovalent cation concentration there is a strong dependence of the hydrodynamic parameters upon salt which is reduced approx. 3-fold at higher salt concentrations. We attribute the compaction of single stranded DNA by salt to electrostatic shielding and, in case of native single stranded DNA, secondary structure formation. Internal motions of the native single stranded DNA are observable at all salt concentrations and can be interpreted with a model of segmental diffusion of the elements of the polymer chain. The observed segmental diffusion coefficient of the native single stranded polynucleotide increases with increasing salt under the conditions investigated.     

Physical properties of type i collagen in solution: Structure of α-Chains and β- and γ-components and two-component mixtures

The structure of thermally denatured Type I collagen has been studied using laser light scattering. The results indicate that the diffusion coefficients of α-chains and β- and γ-components are 1.550 ± 0.08 × 10^?7, 1.000 ± 0.05 × 10^?7, and 0.835 ± 0.04 × 10^?7 cm²/sec, respectively, at temperatures between 20 and 40°C. It is concluded from diffusion data that these species have hydrodynamic radii of about 13.8 nm (α-chain), 21.5 nm (β-component), and 25.7 nm (γ-component), consistent with previous studies of thermal denaturation by light scattering. It is also concluded, based on volume calculations, that a large volume increase occurs when the triple helix unfolds. Homodyne correlation functions for two component mixtures of α-chains and β-and γ-components appeared to decay exponentially. In all but one case discussed the correlation function could be fitted with a single component having a translational diffusion coefficient which was an intensity weighted average of the diffusion coefficient of each component present.     

Dynamic light scattering from collagen solutions. I. Translational diffusion coefficient and aggregation effects

Solutions of native collagen extracted from rat tail tendons in neutral salt solution have been studied by dynamic light scattering. The spectra obtained are consistent with the presence in solution of both single rod-shaped collagen molecules and aggregates of molecules. No contribution to the spectrum has been detected at any scattering angle from rotational diffusion of single molecules, although a measurable broadening effect is expected at high angles. The translational diffusion coefficient D of single molecules, calculated from the broader spectral component, shows an anomalous dependence on collagen concentration with a maximum value of D_20,w = 8.6 ± 0.2 × 10^?12 m²/sec near the concentration 0.04% by weight. Above 0.05% D falls linearly with increasing concentration and takes the value D _20,w = 8.1 ± 0.2 × 10^?12 m²/sec at 0.064% collagen.     

Hydrodynamic properties of proteoglycan subunit from bovine nasal cartilage. Self-association behavior and interaction with hyaluronate studied by laser light scattering

Measurements of translational diffusion coefficients by quasielastic laser light scattering, sedimentation coefficients, and intrinsic viscosities at zero shear of proteoglycan subunit fraction A1-D1-D1 isolated from bovine nasal septa are reported. Molecular weights and hydrodynamic dimensions are compared with those expected on the basis of structural models previously proposed. Comparison of the concentration dependence of the diffusion coefficient in the presence of NaCl and GdnHCl leads to the conclusion that significant self-association behaviour of subunit occurs in the absence of GdnHCl. In the absence of added salt, anomalous nonlinear concentration dependence of D_t estimated from wide-angle light-scattering experiments is observed. In addition, D_t apparently becomes angle dependent. These results are interpreted in terms of the perturbation of normal translational diffusion of the monomer by strong repulsive intermolecular interactions due to the combined effects of long-range electrostatic forces and macromolecular congestion at higher concentrations. By carrying out experiments at small scattering angles, it is possible to determine D for proteoglycan subunit in the absence of supporting electrolyte. Titration of a dilute solution of subunit with hyaluronic acid results in a sigmoidal behaviour of the Stokes radius, indicating the formation of complexes of higher molecular weight results from the noncovalent association of proteoglycan subunits with hyaluronate. Observation of D_t appears to provide a useful method for studying the proteoglycan subunit–hyaluronate interactions.     

Concentration dependence of proteoglycan diffusion

The mutual diffusion coefficient of the bovine nasal cartilage proteoglycan subunit is found to increase rapidly with increasing concentration and decreasing ionic strength. These results have been obtained by analysis of the boundary relaxation of concentration gradients in the analytical ultracentrifuge by schlieren optics. The diffusion behavior can be understood in terms of the nonideality of the proteoglycan. The magnitude of the nonideality is dominated by charge interactions, whereas the influence of molecular size and associated excluded-volume interactions is small. The concentration dependence of the apparent diffusion coefficient of the proteoglycan subunit from dynamic light scattering was found, in contrast, to decrease with increasing concentration. Computer simulation of the dynamic light scattering suggests that the presence of a small population of aggregates may account for the difference in the two types of diffusion measurement due to their marked influence on the scattering.     

Oxygenation of hemoglobin: Correspondence of crystal and solution properties using diffusion coefficient measurements

Quasi-elastic light scattering has been used to measure the change in the translational diffusion coefficient of hemoglobin upon oxygenation and the difference in the diffusion coefficients of oxy- and methemoglobin. The diffusion coefficients of oxy- and methemoglobin were found to be the same within the experimental accuracy of 0.2%, while the diffusion coefficient of oxyhemoglobin tetramers in solution at 13 mg/ml was found to be 0.8% smaller than that of deoxyhemoglobin at the same concentration, when the reversible dissociation of oxyhemoglobin tetramers into dimers was taken into account. In the limit of zero concentration, the oxyhemoglobin diffusion coefficient was found to be 1.5% ± 1.0% smaller than that of deoxyhemoglobin. This result is in very good agreement with what we predict using atomic coordinates to model the liganded and unliganded hemoglobin molecules as ellipsoids of revolution.     

Comparative studies on the conformational change and aggregation behavior of irradiated carrageenans and agar by dynamic light scattering

The conformational associative properties of kappa-, iota-, and lambda-carrageenan and agar with irradiation dose were studied by dynamic light scattering. The random scission of the carrageenans and agar by gamma irradiation resulted in the formation of polydispersed lower molecular weight fragments. At high doses, the system moves towards uniformity. Conformational change from coil to helix was observed in all carrageenans and agar at doses up to 100 kGy. The conformational change in lambda-carrageenan may be due to the irregular and hybrid structure of this polysaccharide. Only agar and lambda-carrageenan still undergo conformational transition at a high dose of 200 kGy. Gelation is observed for kappa-, iota-carrageenan up to a dose of 50 kGy while gelation is still observed at 100 kGy for agar. Increase in the hydrodynamic radius with decreasing temperatures for the non-irradiated carrageenans follows this order: lambda-carrageenan>kappa-carrageenan>iota-carrageenan. Slight increases in hydrodynamic radius were observed with irradiation.     

Ionic strength effects on macroion diffusion and excess light-scattering intensities of short DNA rods

Quasielastic and static light-scattering measurements were made on DNA isolated from chicken erythrocyte mononucleosomes as a function of ionic strength between 6 × 10^?4 and 1.0M. A transition from single-exponential autocorrelation functions to markedly non-single-exponential decays was observed around 10^?2M ionic strength and was accompanied by a large decrease in the excess light-scattering intensity. Autocorrelation functions recorded below 10^?2M salt were well fit by the sum of two exponential relaxation which differed by as much as 100-fold in time constants. Apparent diffusion coefficients for the fast and slow processes plateaued around 10^?3M with numerical values approximately 10-fold and 1/10, respectively, of the translational diffusion coefficient for mononucleosome DNA at high ionic strength. This behavior is similar to that observed with poly(L -lysine), for which the slow decay has been associated with a transition to an extraordinary phase. The strong and complex salt dependence observed here illustrates potential difficulties in deriving structural information from scattering by polyions at low ionic strength.     

Dynamic light scattering studies of ribonuclease

Dynamic light scattering has been used to measure the translational diffusion coefficients of bovine pancreatic ribonuclease A as functions of temperature and concentration in the presence of 1 M Guanidine-HCl. Data was collected throughout a temperature range including the folding-unfolding transitions. Evidence of a pretransition "swelling" of the protein was observed. Entropy and enthalpy changes upon unfolding were obtained using a two-state model.     

Diagnostic of precipitant for biomacromolecule crystallization by quasi-elastic light-scattering

The translational diffusion coefficient D25,w of hen egg-white lysozyme and concanavalin A from the jack bean is measured in various precipitating agent solutions as a function of salt and protein concentration using quasi-elastic light-scattering. With some precipitants, in undersaturated protein solutions, a protein or salt concentration dependence of the diffusion coefficient of the scatters is observed. It can be correlated with the inability of the protein to crystallize in this precipitant once the solution is supersaturated. These variations of D25,w are interpreted in terms of non-specific interactions and/or aggregation that prevent the protein from making appropriate contacts to form a crystal. With other precipitants known to lead to crystallization, no significant variation of the diffusion coefficient with increasing concentration was observed, indicating that under such conditions up to saturation the proteins remain essentially monodisperse. Application of this technique to find crystallization conditions of other proteins is discussed.     

Effect of aminoacylation on tRNA conformation

Translational diffusion coefficients have been simulated for various conformations of tRNA^Phe (yeast) by bead models, in order to analyze data obtained by dynamic light scattering on the free and the aminoacylated form. The 18% increase of the translational diffusion coefficient upon deacylation, reported by Potts et al. (1981), could not be represented by any change of the L-hinge angle, but could only be simulated by a conformation change to an extended form with extensive dissociation of base pairs. Since extensive unpairing is not consistent with evidence accumulated in the literature, the change of the diffusion coefficient must be mainly due to processes other than intramolecular conformational changes.     

A method for measuring sedimentation and diffusion of macromolecules in capillary tubes by total intensity and quasi-elastic light-scattering techniques.

Light scattered from a macromolecular solution in a capillary tube is used to determine both the sedimentation and translational diffusion coefficients. The capillary tube is spun in a preparative centrifuge, removed, and placed in a light-scattering photometer equipped with a scanning mechanism. The intensity distribution of scattered light along the tube represents the concentration profile in the tube and provides the measure of boundary migration. The sedimentation coefficient is determined from this measure and the applied centrifugal field. The diffusion coefficient is obtained from a time-autocorrelation analysis of fluctuations in intensity of light scattered from any fixed point of the profile. These coefficients were obtained for two monodisperse systems, R17 bacteriophage and 28s ribosomal rat liver RNA. The molecular weights obtained from ratios of these coefficients are in good agreement with literature values. In the sedimentation analysis, deviations from linearity between boundary displacement and applied field were found to be less than 1%. This precision confirms that the boundary is stable for the capillary geometry even in the absence of a preformed density gradient. The sedimentation coefficients of identical samples were also measured with the Spinco Model E analytical ultracentrifuge; results of the two methods agree to within 4%. As a consequence of the capillary tube geometry and light-scattering detection, sedimentation coefficients can be obtained from sample volumes of less than 100 μl. This detection techniques is thus far demonstrated to be at least an order of magnitude more sensitive than Schlieren optics, thereby useful when uv absorption is not applicable. For diffusion measurements there are also several inherent advantages. The diffusion coefficient is obtained from the identical sample, and scanning provides the capability to measure D from various parts of the sedimentation profiles and thereby directly explore concentration dependence, homogeneity, and integrity of the sample. The capillary tube with a layer of silicone oil over the sample and centrifugation provides an effective method to cleanse the solution and trap all dust.     

A rotational diffusion coefficient of the 70S ribosome determined by depolarized laser light scattering

We have obtained a rotational diffusion coefficient of the 70S ribosome isolated from Escherichia-coli (MRE-600), from the depolarized light scattering spectrum measured by photon correlation spectroscopy. The intensity correlation function of depolarized scattered light contains contributions due to multiple scattered and anisotropy scattered light from the ribosomal particle. We discuss extensively the subtraction procedure used to obtain the rotational correlation time from the experimental correlation function. We have also obtained the translational diffusion coefficient from the same sample by determining the polarized correlation function. The hydrodynamic radius determined from the rotational diffusion coefficient is only slightly larger than the radius obtained from the translational diffusion coefficient. Therefore the ribosomal particle has a non-spherical shape. This conclusion, however, could be impaired by the effect of free draining of the ribosome.     

Dynamic light scattering from solutions of microtubules.

Calf brain microtubule protein was assembled in vitro to form dilute solutions of microtubules (240 A diameter) having lengths greater than 1 micrometer. The microtubule solutions were examined by dynamic laser light scattering techniques. The angular dependence of the correlation function leads to the conclusion that the correlation function is measuring the translational diffusion constant of the particles. The length dependence of the correlation function, however, shows that the translational diffusion constant is not being measured and that the diffusion constant for the microtubules cannot be straightforwardly determined. These results suggest that a collective property of the rods is being measured by the laser light scattering. Although specific microtubule-microtubule interactions are a possible explanation for the observed results, we present arguments that suggest that the solution can be adequately modeled as a network of entangled polymers.     

A rotational diffusion coefficient of the 70S ribosome determined by depolarized laser light scattering.

We have obtained a rotational diffusion coefficient of the 70S ribosome isolated from Escherichia-coli (MRE-600), from the depolarized light scattering spectrum measured by photon correlation spectroscopy. The intensity correlation function of depolarized scattered light contains contributions due to multiple scattered and anisotropy scattered light from the ribosomal particle. We discuss extensively the subtraction procedure used to obtain the rotational correlation from the time from the experimental correlation function. We have also obtained the translational diffusion coefficient from the same sample by determining the polarized correlation function. The hydrodynamic radius determined from the rotational diffusion coefficient is only slightly larger than the radius obtained from the translational diffusion coefficient. Therefore the ribosomal particle has a non-spherical shape. This conclusion, however, could be impaired by the effect of free draining of the ribosome.     

Dynamic light scattering measurements of the diffusion of probes in filamentous actin solutions

The diffusion coefficients of monodisperse polystyrene latex spheres in solutions of polymerized actin were measured using dynamic light scattering. Four different probes with radii R, ranging from 50 to 500 nm, were separately used in actin solutions with concentrations c, ranging from 1.5 to 21 microM, which had been polymerized with either 1 mM MgCl2, 1 mM CaCl2, or 100 mM KCl. Under all conditions, and at four different scattering angles in the range of 30 degrees-90 degrees, the measured average diffusion coefficients D of the probes were systematically smaller for samples of increased actin concentration or of increased probe radius. Control experiments indicated that the probes did not bind to the actin. These data for Mg2+- and Ca2+-polymerized actin agree and were found to be quite well summarized by the scaling relation D/D0 = exp[-alpha R delta c nu], where D0 is the measured diffusion coefficient of the probes in water (and, as also measured, in the starting actin solutions prior to polymerization with added salt), with values of delta = 0.73 +/- 0.05, nu = 1.08 +/- 0.09, and alpha = (1.1 +/- 0.6) x 10(-3) (with c in microM and R in nm). Data for KCl-polymerized actin show much more restricted diffusivities of the probes at comparable actin concentrations. Inhomogeneities in the solution are reflected in the "effective polydispersity" of the probe diffusion coefficients, which depend on local microviscosity differences.     

Intensity-fluctuation spectroscopy and tRNA conformation. II. Changes of size and shape of tRNA in the melting process

We have measured the translational (D^T) and rotational (D^R) diffusion coefficients of bulk tRNA from baker's yeast during the thermal unfolding process by means of photon-correlation spectroscopy. It should be noted that our estimate of the rotational diffusion coefficient represented, for the first time, measurements on a small macromolecule in solution by the photoelectron time-of-arrival technique with a delay-time resolution of 1 nsec. The melting curves expressed in terms of δD^T vs temperature were consistent with the literature data in revealing the melting steps and their dependence on NaCl concentration. Additionally, it was possible to prove the existence of an intermediate, more compact structure during the initial steps of the thermal unfolding process. We found that the temperature ranges over which this intermediate structure appears depend strongly on salt concentration. By utilizing both translational and rotational diffusion coefficients and Perrin's equations for ellipsoids of revolution, we have computed the values of the equivalent length and width of tRNA molecules in solution at four different temperatures for NaCl concentrations of 0.2, 0.5, and 1M. The approximate model of ellipsoids of revolution also permits us to obtain an estimate of the radius of gyration, which is in very good agreement with literature data measured by means of small-angle x-ray scattering. Furthermore, we have measured the shape and size changes of tRNA with varying NaCl concentrations at room temperatures (25°C). The molecule becomes smaller and more spherical when NaCl concentration increases. As a result of partial melting at 70°C, the macromolecule is surprisingly elongated with an approximate axial ratio of 8:1 and has dimensions of about 180/22Å. Such information on conformational changes by a simultaneous determination of rotational and translational diffusion coefficients illustrates the potential of this approach, not available by other methods.