Intensity fluctuation spectroscopy and transfer RNA conformation. III. Influence of NaCl concentration on the size and shape of the initially salt-free tRNA in solution.

The influence of sodium ion concentration in solution on the initially salt-free conformation of bulk tRNA from baker's yeast has been investigated by means of photon correlation spectroscopy. From the measured values of translational (D^T) and rotational (D^R) diffusion coefficients, the semiaxes of an ellipsoid of revolution, which are hydrodynamically equivalent to the tRNA molecule, were calculated for tRNA solutions in pure H₂O as well as in 0.005, 0.1, 0.5M NaCl and 0.01M MgCl₂ solutions at pH 4.2 and 7.5. These data, combined with our previous studies, suggested a model which describes the formation of an ordered tRNA structure due to increasing NaCl concentrations. Furthermore, we have obtained information concerning intermolecular interactions between tRNA molecules in solution. In low-salt or salt-free tRNA solutions, we detected in the linewidth distribution function an extra-fast component which can be attributed as possibly due to charge fluctuations related to the reaction of ionization of organic bases. In our light-scattering linewidth measurements, we do not see fluctuations of charged and uncharged states directly as concentration fluctuations. Rather, we postulate a modulation of long-range intermolecular electrostatic interactions between the tRNA molecules due to such charge fluctuations. It is this modulation which is related to the fast component of the time correlation function at finite concentrations. A quantitative theory is needed to provide a more definitive explanation of the dynamical behavior of tRNA in salt-free or low-salt solutions.     

Translational and rotational diffusion of tobacco mosaic virus from polarized and depolarized light scattering

The translational and rotational dynamics of tobacco mosaic virus in sodium phosphate buffer (pH =7.5) solutions has been investigated by polarized and depolarized light scattering Rayleigh linewidth studies. For concentrations ranging from 1.75 × 10^?4 g ml^?1 to 0.25 × 10^?4 g ml^?1 the translational diffusion coefficient (D_T) has been found to be slightly concentration dependent and extrapolation to zero concentration gives D₀^20°C = 0.34 ± 0.01 × 10^?7 cm²S^?1. A full analysis of the polarized spectra obtained at high and low scattering angles and the depolarized spectra at near zero scattering angles has enabled these techniques to be compared and the rotational diffusion constant D_R to be determined. At a solution concentration of 1.75 × 10^?4 g ml^?1 a mean value is found to be D_R^20°C = 350 ± 30s^?1. These values of D_T and D_R are in approximate agreement with calculations based on models of the tobacco mosaic virus molecule as a cylindrical rod.     

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.     

Transient electric birefringence of T-even bacteriophages. I. T4B in the absence of tryptophan and fiberless T4 particles

Measurements of the electric birefringence of suspensions of T4B in the absence of tryptophan and of fiberless T4 particles show that both kinds of particles are hydrodynamically equivalent. Their rotational diffusion coefficients corrected to 25°C and water viscosity (D_25,w) are 280 ± 9 sec^?1 and 295 ± 10 sec^?1, respectively. These corrected rotational diffusion coefficients are almost independent of buffer concentration and temperature. The sedimentation coefficient (s_20,w) of T4 B is equal to 1023 ± 12 S, a value which is likewise independent of buffer concentration. By analysis of the field strength dependence of the steady-state birefringence and by reversing pulse experiments it could be shown that the orientation in an electric field is largely due to a permanent dipole moment. This dipole moment is somewhat dependent on buffer concentration and amounts to about 24,000 debye for T4B and 95,000 debye for fiberless T4. An approximate calculation shows that the difference in dipole moment may be ascribed to positive charges on the fiber tip (at least ten per fiber), to negative charges along the fiber or (and) positive charges on the fiberless particle at those places where the fibers are attached in normal particles.     

Hydrodynamic properties of macromolecular complexes. V. Improved calculation of rotational diffusion coefficient and intrinsic viscosity

In our previous calculations of rotational diffusion coefficients and intrinsic viscosities of macromolecular complexes modeled by arrays of spherical subunits [J. G. de la Torre & V. A. Bloomfield, Biopolymers 16 , 1765, 1779 (1977); 17 , 1605 (1978)], results were poor when the dominant subunit was located near the center of frictional resistance. A simple means of correcting this flaw, which gives satisfactorily accurate results with little increase in computation time, is to replace the single large subunit with an array of smaller ones. We have examined trigonal bipyramidal, octahedral, and cubic arrays of spheres whose radii were chosen to give the same total volume or the same rotational diffusion coefficient as the parent sphere. These all give similar results, so the details of the modeling are not important. Results obtained using this stratagem are in much better agreement with the theories of Perrin and Simha for short prolate ellipsoids of revolution, and with experimental measurements of rotational diffusion coefficients of T-even bacteriophage without fibers or with fibers retracted. We have also extended our previous calculations to consider phage with various numbers of fibers attached.     

A Numerical Inversion of the Perrin Equations for Rotational Diffusion Constants for Ellipsoids of Revolution by Iterative Techniques

The rotational diffusion coefficients R₁ and R₃ for ellipsoids of revolution are shown to represent another pair of hydrodynamic data to obtain size and shape with theories by Sadron and Scheraga-Mandelkern. An iterative numerical technique is presented which allows the semiaxes to be determined from the Perrin equations for rotational diffusion constants. The use of this inversion technique is illustrated by application to literature data from dielectric dispersion studies.     

Dependence of laser light scattering of DNA on NaCl concentration

We show that the persistence length a of DNA, derived from total intensity laser light scattering of linear Col E₁ DNA and corrected for excluded-volume effects, varies from about 68 nm in 0.005M NaCl to about 40 nm in 0.2M NaCl, leveling off to a constant value (about 27 nm) at high NaCl (1–4M) concentration. These observations do not agree with current views on the effect of electrostatic charge and ionic conditions on DNA dimensions. The apparent diffusion constant D_app, determined from laser light scattering autocorrelation as a function of scattering vector q, at NaCl concentrations 0.005–4M, correctly yields the translational diffusion coefficient D_t at low values of q and scales with molecular dimensions rather than segment length at high values of q; thus, D_app/D_t yields a universal curve when plotted against q²R, where R_g is the radius of the gyration. The sedimentation coefficients s at 0.1 and 0.2M NaCl concentration closely agree with the well-tested empirical relations, and a combination of s, D_t, and the appropriate density increments yield correct molar masses over the whole salt concentration range. Approximate constancy of D_tR_g indicates limited draining in translational flow. We present some observations and thoughts on the regimes in which a dependence of the correlation decay times on q³ rather than q² applies. We conclude that quasielastic laser light scattering discloses little information about dynamics of internal motion of DNA chains.     

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.     

Histone-induced conformational changes in DNA as probed by quasi-elastic light scattering.

Quasi-elastic light scattering as measured by intensity fluctuation (self-beat) spectroscopy in the time domain can be profitably used to follow both the translational diffusion D and the dominant internal flexing mode τ_int of DNA and its complexes with various histones in aqueous salt solutions. Without histones, DNA is found to have D = 1.6 × 10^?8 cm²/sec and τ_int ? 5 × 10^?4 sec in 0.8 M NaCl, 2 M urea at 20°C. Total histone as well as fraction F2A induce supercoiling (D = 2.6 × 10^?8 cm²/sec, τ_int ? 2.8 × 10^?4 sec) whereas fraction F1 induces uncoiling (D = 1.0 × 10^?8 cm²/sec, τ_int ? 9.4 × 10^?4 sec). Upon increasing the salt concentration to 1.5 M the DNA–histone complex dissociates (D = 1.8 × 10^?8 cm²/sec). Upon decreasing the salt concentration to far below 0.8 M, the DNA–histone complex eventually precipitates as a chromatin gel.     

Quasielastic light-scattering studies on human fibrinogen and fibrin. II. Fibrin polymerization

The shape of fibrin intermediate polymers as well as the rate of fibrinogen polymerization was studied using diffusion measured by quasielastic light scattering. After their length distribution was narrowed by gel filtration, the polymers yielded translational and rotational diffusion coefficients of 0.37 ± 0.05 × 10^?7 cm² sec^?1 and 142 ± 32 sec^?1, respectively. Theoretical considerations indicated the polymers to be rigid rods. The rate of polymerization of fibrinogen monitored by diffusion paralleled that provided by simulataneous intensity measurements. Both monitors indicated polymerization occurs most rapidly at 30°C.     

Cation-induced conformational changes in tRNA molecules

The uv absorption spectra and melting profiles of an initially ion-free solution of E. coli unfractionated tRNA are significantly modified by the addition of either Na⁺, Mg²⁺, or Mn²⁺ or of other first-series transition-metal ions such as Ni²⁺, Co²⁺, and Zn²⁺. The main effect of the addition of all monovalent or divalent cations examined is an increase of the ordered and stacking stabilized tRNA structure, as revealed by a drop in the absorption near 260 nm, as well as in the 4-TU absorption region. Sharp differences have, however, been detected in the 290–305-nm range in the presence of the various ions studied. When transition-metal ions were added to a tRNA solution, an absorption peak appeared at 294 nm. This effect is interpreted as a perturbation of the electronic structure of the bases due to direct binding of metal ions to the bases. An analysis of the variation in the spectrum as a function of metal concentration and of the thermal melting reversibility in the presence of various metal ions supports the conclusion that while all ions investigated are involved in binding to the phosphate groups of tRNA, transition-metal ions are also able to bind directly to the bases.     

tRNA structure and binding sites for cations

Equilibrium dialysis and electronic and nuclear resonance spectroscopy show that tRNA cooperatively binds divalent metal ions at very low concentrations (free metal concentration 3 × 10 ^?6 M). The first two methods show that different purified tRNAs have a very similar behavior, including initiator tRNA_F^met. tRNAs with an extra arm in the clover-leaf model, however, appear to have a slightly different behavior. The binding can be described in terms of two classes of sites. The cooperative association of divalent ions binding first does not parallel a cooperative change in the hyperchromism of the tRNA, while the non-cooperative association of the second class of divalent ions corresponds to the concentrations needed to obtain a cooperative melting of the tRNA. The temperature dependence of the number of binding sites and of their binding constants is also presented. The nature of the divalent ion gives the following efficiency: for the cooperativity Co⁺⁺>Mg⁺⁺>Mn⁺⁺ for the weak binding sites Mn⁺⁺>Co⁺⁺>Mg⁺⁺     

Thermodynamics of transfer ribonucleic acids: the effect of sodium on the thermal unfolding of yeast tRNAPhe.

The thermal unfolding of yeast phenylalanine-specific tRNA (tRNA^Phe) has been calorimetrically investigated at several salt concentrations in the absence of magnesium. Application of the deconvolution theory of macromolecular conformational transitions allows calculation of the thermodynamic parameters of unfolding. It is demonstrated that the unfolding of tRNA^Phe occurs in a sequential fashion and that four separate transitions or five macromolecular thermodynamic states exist in the temperature range 8–72°C under the experimental conditions of these studies (0.067–0.52M Na⁺). The enthalpy and entropy changes between states and the relative population of each state as a function of temperature and salt concentration have been obtained. Sodium stabilizes the low-temperature conformations of tRNA^Phe. The increase in the melting temperatures of each transition is shown to be linearly dependent on the logarithm of sodium concentration. These results allow calculation of the “phase” diagram for the transitions as a function of salt concentration.     

Quasielastic light scattering from solutions of filamentous viruses. I. Experimental

Intensity fluctuations of laser light scattered from filamentous viruses Pf1 [length L (Å) × diameter d (Å) = 20,000 × 90], M13 (9000 × 90), potato virus X (5150 × 130), and tobacco mosaic virus (3000 × 180) in sucrose density gradients were measured with a photon correlation spectrometer over a range of scattering angles from 15° to 120°. The experimental data can be approximated by two exponential decays, “slow” and “fast.” The slow decay rate constant t corresponds to the translational diffusion D of the virus, i.e., t = K²D, where K is the magnitude of the scattering vector. The amplitude of the slow component, i.e., translational diffusion, remains greater than that of the fast component, even at high KL. The fast decay rate constant t is also proportional to K² for viruses such as Pf1, M13, and even potato virus X. In the companion paper, we shall attribute the amplitude enhancement of the translational diffusion to the coupling of its anisotropy to the rotational diffusion modes. In order to explain the excessive decay rates in the fast component, we need to consider the bending mode of rodlike viruses, especially in the longer viruses such as M13 and Pf1, in addition to the usually expected rotational diffusion modes.     

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.     

Studies of Ribosomal Diffusion Coefficients Using Laser Light-Scattering Spectroscopy

Using an optical beating technique, the diffusion coefficients and relative scattered intensity of Escherichia coli 70S, 50S, and 30S ribosomes are measured as a function of temperature and Mg²⁺ concentration. For solutions at 10 mM Mg²⁺ and between 0°C and about 40°C, the values of D_20,w obtained are 1.7, 1.9, and ≈2.1 × 10^-7 cm²/s, respectively. Preparative procedures drastically affect these values and equivalent hydrodynamic ellipsoids of revolution models give large axial ratios indicating extensive hydration or a deviation from the assumed shape. Calculations also indicate that the subunits expand upon dissociation. Measurements of D_20,w vs. temperature indicate that 70S particles undergo a conformational change prior to dissociation and can be heat dissociated at 30-32°C at low concentrations. Treatment of 70S ribosomes with EDTA causes a biphasic dissociation reaction. Addition of Mg²⁺ after dissociation with EDTA shows that longer waiting times yield fewer 70S particles and that even short waiting times may yield ribosomes differing from the native conformation. Addition of p-chloromercuribenzoic acid (PCMB) is shown to dissociate 70S particles, but to a lesser extent than ethylenediaminetetraacetic acid (EDTA).     

Static and dynamic light scattering from dilute insulin solutions

Static and dynamic light-scattering measurements are reported on zinc-insulin at room temperature (21 ± l°C) and pH = 6.88 in 0.1M NaCl aqueous solution. The experiments were performed at very low concentration, in the range 0.12 × 10^?4 to 0.90 × 10^?4 g cm^?3. Within experimental error, we find no evidence for a critical micellar concentration in this system. The aggregation phenomenon starts immediately after preparation of the solutions, and takes several days to come to stable equilibrium. The concentration dependence of the diffusion coefficients, D _z, = D_o (1 — k_DC), is negative, and k_D was observed to decrease as a function of time, while the aggregate size was found to increase. The equivalent concentration coefficient, ?2BM _W, obtained from static light scattering, showed a similar behavior, and, within experimental error, was found to be numerically equal to k_D. From the relation found between the diffusion coefficient at infinite dilution and the molecular weight of the aggregates, log D₀ = ?0.240 log M _w ? 5.077, we deduce that the insulin aggregates are compact structures with a characteristic radius of 0.71 Å/(dalton)^1/3, surrounded by a hydration layer of a thickness of 8.0 Å. The equilibrium aggregation number is approximately 10.     

Rotational diffusion coefficients of a small,spherical subunit flexibly tethered to a larger sphere

The rotational diffusion coefficients of a small spherical particle, which is flexibly anchored to the surface of a much larger sphere, are calculated using the hydrodynamic theory of segmentally flexible particles. The model is intended for representing the rotational mobility of a small residue or chromophore in the surface of a globular macromolecule. The coefficients are found to be essentially independent, or to vary slowly with the relative dispositions of the spheres. They are also insensitive to the size ratio when this ratio is high enough. These findings support the use of an approximative treatment proposed by Wegener in which the small conformation dependence is averaged out. The resulting averages are tentatively used in the Lipari-Szabo model for restricted rotational diffusion in a cone. It is concluded that the rotational relaxation of the small sphere has three components: (i) a torsional rotation with the same diffusion coefficient as the free sphere; (ii) a perpendicular wobbling with a diffusion coefficient several (five in a typical case) times smaller; and (iii) an overall rotation of the whole macromolecule, that will appear in a much longer time scale if the two spheres have quite distinct sizes.