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.     

Influence of double scattering in determination of rotational diffusion coefficients by depolarized dynamic light scattering: Application to the bacteriophages T7 and T4B

Dynamic light scattering experiments were performed on solutions of the bacteriophages T7 and T4B in order to obtain the rotational diffusion coefficients of these phages. Correlation functions were determined from the depolarized intensity scattered in the forward direction. The apparatus used in this study is described in detail. Particular attention is paid to the minimalization of the depolarized intensity due to double scattering. If double scattering cannot be neglected, the correlation function of the depolarized field is the sum of the correlation functions resulting from single and double scattering. It is shown that by correcting for double scattering, it is then possible to obtain the rotational diffusion coefficient of the macromolecules. Although the optical anisotropy of both T4B (retracted fibers) and T7 is very small, the experimental conditions could be chosen in such a way that no depolarized scattering due to double scattering was observed. The measured rotational diffusion coefficients for T4B and T7 are D = 258 ± 12 and 4528 ± 100 sec^?1, respectively. These values compare very well with those obtained by electric birefringence experiments.     

Translational and Rotational Diffusion Constants of Tobacco Mosaic Virus from Rayleigh Linewidths

The translational and rotational diffusion constants of tobacco mosaic virus (TMV) have been determined from homodyne and heterodyne measurements of the spectrum of laser light scattered from dilute aqueous solutions of TMV. Our results for the translational and rotational constants respectively, reduced to 20 degrees C, are: D(T) = 0.280 +/- 0.006 x 10(-7) cm(2)/sec, and D(R) = 320 +/- 18 sec(-1). We include a theoretical derivation of the spectrum of light scattered from rod-shaped molecules which reproduces results obtained previously by Pecora, but which is specialized at the outset to the problem of dilute solutions so that simple single-particle correlation functions may be utilized. An analysis of the photocurrent spectrum for both the homodyne and heterodyne detection schemes is given. Various data reduction schemes utilized in the analysis of our spectra are described in some detail, and our results are compared with values of the diffusion constants obtained from other experiments.     

Observation of flagellation of spermatozoa by depolarized laser light scattering.

Depolarized laser light-scattering theory was applied to derive the autocorrelation function of laser light scattered by motile spermatozoa, assuming that each spermatozoon is a chain of rotatable rigid ellipsoids of revolution and also that the rotational velocity about an axis perpendicular to the symmetry axis of the ellipsoid is constant for times of the order of the characteristic decay time of the autocorrelation function. The rotations are produced by flagellar movements of the spermatozoa. The correlation function thus obtained was related to the second-order coefficient of a Legendre polynomial expansion of the rotation of the direction angle of the ellipsoidal axis. The experimental fact that the correlation function for dead spermatozoa of sea urchin resembled that for flagella mechanically separated from spermatozoa indicated to us that the depolarized light was scattered mainly by flagella. The rotational velocity distribution of the flagella was determined by comparing the theoretical analysis with the experimentally obtained correlation functions for the motile and dead spermatozoa. The value of the average velocity caused by the flagellation, 230 rad/s, was in good agreement with that measured under an optical microscope.     

Diffusive properties of water in Artemia cysts as determined from quasi-elastic neutron scattering spectra.

Results have been obtained on the quasi-elastic spectra of neutrons scattered from pure water, a 20% agarose gel (hydration four grams H2O per gram of dry solid) and cysts of the brine shrimp Artemia for hydrations between 0.10 and 1.2 grams H2O per gram of dry solids. The spectra were interpreted using a two-component model that included contributions from the covalently bonded protons and the hydration water, and a mobile water fraction. The mobile fraction was described by a jump-diffusion correlation function for the translation motion and a simple diffusive orientational correlation function. The results for the line widths gamma (Q2) for pure water were in good agreement with previous measurements. The agarose results were consistent with NMR measurements that show a slightly reduced translational diffusion for the mobile water fraction. The Artemia results show that the translational diffusion coefficient of the mobile water fraction was greatly reduced from that of pure water. The line width was determined mainly by the rotational motion, which was also substantially reduced from the pure water value as determined from dielectric relaxation studies. The translational and rotational diffusion parameters were consistent with the NMR measurements of diffusion and relaxation. Values for the hydration fraction and the mean square thermal displacement [u2] as determined from the Q-dependence of the line areas were also obtained.     

Dynamic light scattering from dilute suspensions of thin discs and thin rods as limiting forms of cylinder, ellipsoid and ellipsoidal shell of revolution

A previous formulation of the field correlation function G1(tau) of light quasielastically scattered from suspensions of rigid rods undergoing anisotropic translational as well as rotational diffusion (T. Maeda and S. Fujime, Macromolecules 17 (1984) 1157) was extended to the cases of suspensions of cylinders (length L and radius R), ellipsoids and ellipsoidal shells of revolution (x2/b2 + y2/b2 + z2/a2 = 1). The present formulation includes that for suspensions of rigid rods in the limit of KR 1 or in the limit of b/a 1 and Kb 1 (an extremely prolate ellipsoid), and also that for suspensions of discs in the limit of KL 1 or in the limit of b/a 1 and Ka 1 (an extremely oblate ellipsoid), where K is the length of the scattering vector. Explicit forms of G1(tau), of the first cumulant Gamma of G1(tau) and of the dynamic form factors will be given, and numerical methods suitable for computation of dynamic form factors will be discussed. The present results can be applied to the analysis of experimental data for dilute suspensions of thin rods and thin discs. When the situation is favorable, our method can provide transport coefficients D1, D3, and Theta from dynamic light-scattering data only, where D(1) and D(3) are, respectively, the translational diffusion coefficients parallel with the x (y) and z axes, and Theta the rotational diffusion coefficient around the x (y) axis.     

Hydrodynamic properties of a double-helical model for DNA.

The translational and rotational diffusion coefficients of very short DNA fragments have been calculated using a double-helical bead model in which each nucleotide is represented by one bead. The radius of the helix is regarded as an adjustable parameter. The translational coefficient and the perpendicular rotation coefficient agree very well with experimental values for oligonuclotides with 8, 12, and 20 base pairs, for a single value of the helical radius of about 10 A. We have also calculated a nuclear magnetic resonance relaxation time in which the coefficient for rotation about the main axis is involved. As found previously with cylindrical models, the results deviate from experimental values, indicating that the internal motion of the bases has a remarkable amplitude. An attempt to quantify the extent of internal motions is presented.     

Quasi-elastic light scattering of Artemia ribosomes sedimented in a CsCl density gradient

It is impossible to measure the diffusion coefficient of macromolecules directly and accurately by quasi—elastic light scattering, when aggregates cannot be eliminated from the solutions to be investigated. Nevertheless, a simple method can be applied to overcome this problem in many cases. Aggregates are separated from the monomeric macromolecules by rate-zonal sedimentation in a CsCl density gradient in a transparent centrifugation tube; the monomers are then located by laser light scattering intensity measurements; photon correlation spectroscopy of the scattered light finally yields their diffusion coefficient. The viscosity of aqueous CsCl solutions at different temperatures and concentrations allows a good separation by centrifugation and a low uncertainty in the reduction of the measured diffusion coefficient to standard conditions.The application of the method to eukaryotic large ribosomal subunits is described as an example.     

Rod-like cholesterol micelles in aqueous solution studied using polarized and depolarized dynamic light scattering.

Micelles of cholesterol in aqueous solution have been investigated using polarized and depolarized dynamic light scattering. They are shown to be highly extended and characterized by a narrow size distribution. It is shown that a rod-like model is applicable with length, L = 580 nm. Determination of the rotational diffusion coefficient by analysis of the autocorrelation function gave a value of theta = 150 s-1, which is close to the calculated value for the rod with this dimension. Depolarized dynamic light scattering measurements as a function of angle gave a value of 110 s-1.     

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.     

Quasielastic light scattering from solutions of filamentous viruses. II. Comparison between theories and experiments

We have compared four theoretical effects of rodlike macromolecules with the fast components, i.e., components other than translational diffusion, of our experimental data, which are presented as amplitude autocorrelation functions of electric field scattered from dilute solutions of monodisperse rodlike viruses with lengths from 3300 Å for tobacco mosaic virus to 20,000 Å for Pf1. The four effects are (1) the optic anisotropy treated by Aragón and Pecora, (2) coupled translational–rotational diffusion due to anisotropy in translational mobility recently reformulated by Gierke, (3) anisotropic rotational diffusion with respect to the direction of translational displacement first discussed by Berne and Pecora, and (4) the bending mode of a rod by Fujime and Maruyama. We show that both the first and second effects are required to explain the enhancement of amplitude of the translational diffusion at the expense of fast components. The experimental decay rates of the fast component exceed that of the rotational diffusions. In order to explain the excessive decay rate in the fast component, we need to include a minute amount (～1%) of bending mode of rodlike viruses, especially in longer viruses such as M13 and Pf1.     

Structural analysis of G-DNA in solution: A combination of polarized and depolarized dynamic light scattering with hydrodynamic model calculations.

Specific intra- and intermolecular quadruplex conformations of model G-DNA oligonucleotides have been identified from their translational and rotational diffusion coefficients in aqueous solution. The transport properties were determined by polarized and depolarized dynamic light scattering. A comparison with hydrodynamic model calculations provides detailed information about the size and shape of the molecules and allows one to distinguish between alternative intra- and intermolecular association. The potential of this combination of methods to elucidate biomolecular structures in solution, to characterize conformational changes, and follow intermolecular interaction processes due to a response to external stimuli has been discussed.     

Rotational relaxation of macromolecules determined by dynamic light scattering. II. Temperature dependence for DNA

Correlation functions have been determined for the fluctuating intensity of the depolarized component of forward-scattered laser light from solutions of DNA. The molecular correlation function of calf thymus DNA (mol wt ～15 × 10⁶) appears to exhibit a longest relaxation time (τ_25,w, ～ 18 msec) close to what one would predict from the flowdichroism measurements of Callis and Davidson and, in addition, manifests a spectrum of faster times down to tenths of milliseconds. Furthermore, a major fraction of the amplitude of fluctuations in the angular distribution of segment axes is relaxed on a very much shorter time scale (of the order of 20 microseconds) that appears to be relatively insensitive to molecular weight of the DNA, or to near-melting temperatures. The temperature profile of the longest relaxation time has been obtained and found to exhibit a peculiar spike near T_m, which, together with the absence of a corresponding spike in the (high shear) viscosity, has been interpreted as indicative of an increase in the molecular weight of the DNA in a narrow temperature region near T_m. Correlation functions for polarized light scattered at finite angles were obtained in an attempt to determine the temperature dependence of the translational diffusion coefficient. Although the data contain an extremely slow component that does not admit a simple interpretation, there is some indication of a decrease in the translational diffusion coefficient near T_m, thus supporting the notion of an aggregation occurring near T_m. Finally, a “counterion escape” mechanisn is proposed for the apparent aggregation.     

Dynamic light scattering from collagen solutions. II. Photon correlation study of the depolarized light.

The depolarized forward-scattered light from solutions of rat tail collagen has been studied by photon correlation spectroscopy. The measured autocorrelation function is seen to decay on two widely different time scales. The decay time for the fast component is consistent with the rotational diffusion of rodlike collagen monomers. The slowly decaying autocorrelation component is attributed to large nonspecific aggregates of collagen. A substantial fraction of the collagen is in this aggregated form. Extrapolation of the faster decay times to zero concentration yields a value of θ = 1082 ± 30 sec^?1 for the rotational diffusion coefficient of the collagen monomer.     

Dynamics of Deinococcus radiodurans under controlled growth conditions

Deinococcus radiodurans is a potent radiation resistant bacterium with immense potential in nuclear waste treatment. In this investigation, the translational and rotational dynamics of dilute suspensions of D. radiodurans cultured under controlled growth conditions was studied by the polarized and depolarized dynamic light-scattering (DLS) techniques. Additionally, confocal laser scanning microscopy was used for characterizing the cultured samples and also for identification of D. radiodurans dimer, tetramer, and multimer morphologies. The data obtained showed translational diffusion coefficients (DT) of 1.2 x 10(-9), 1.97 x 10(-9), and 2.12 x 10(-9) cm2 /s, corresponding to an average size of 3.61, 2.22, and 2.06 microm, respectively, for live multimer, tetramer, and dimer forms of D. radiodurans. Depolarized DLS experiments showed very slow rotational diffusion coefficients (DR) of 0.182/s for dimer and 0.098/s for tetramer morphologies. No measurable rotational diffusion was observed for multimer form. Polarized DLS measurements on live D. radiodurans confirmed that the bacterium is nonmotile in nature. The dynamics of the dead dimer and tetramer D. radiodurans were also studied using polarized and depolarized DLS experiments and compared with the dynamics of live species. The dead cells were slightly smaller in size when compared to the live cells. However, no additional information could be obtained for dead cells from the polarized and depolarized dynamic light-scattering studies.     

Conformational studies of BSA using laser light scattering

The usefulness of laser light scattering as a technique for determining protein conformation has been investigated by studying the self-association and drug binding of bovine serum albumin (BSA). The diffusion coefficients of BSA monomers and dimers have been measured and the ratio of these two quantities indicates that in the dimer, the subunit separation is 2.2 times the monomeric hydrodynamic radius. The binding of salicylate to BSA causes an increase in its diffusion coefficient corresponding to a reduction in the frictional drag of the solvent on the protein molecules. It has been found that data obtained using laser light scattering may be interpreted confidently only when proper care has been taken in sample preparation and the scattered intensity autocorrelation function has been appropriately analyzed.     

Theory of light scattering from hollow spheres

Light-scattering from a dilute, fluid disperson of rigid, hollow spheres is treated in the Rayleigh—Debye approximation. The hollow spheres are composed of cylindrically symmetric, radially oriented scattering elements. Analytic expressions for all form factors are derived. The time correlation function of the scattered intensity depends only on the translational diffusion coefficient of the sphere.     

RNA-protein distance patterns in ribosomes reveal the mechanism of translational attenuation

Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.     

Structural similarity of E. coli 5S rRNA in solution and within the ribosome

The article presents translational and rotational diffusion coefficients of 5S rRNA determined experimentally by the method of dynamic light scattering (DLS) and its comparison with the values predicted for different models of this molecule. The tertiary structure of free 5S rRNA was proposed on the basis of the atomic structures of the 5S rRNA from E. coli and H. marismortui extracted from the ribosome. A comparison of the values of DT, tauR, and Rg predicted for different models with experimental results for the free molecule in solution suggests that free 5S rRNA is less compact than that in the complex with ribosomal proteins. In general, the molecules of 5S rRNA consist of three domains: a short one and two longer ones. As follows from a comparison of the results of our simulations with experimental values, in the molecule in solution the two closest helical fragments of the longer domains remain collinear, whereas the short domain takes a position significantly deviated from them.     

Association of protein biogenesis factors at the yeast ribosomal tunnel exit is affected by the translational status and nascent polypeptide sequence

Ribosome-associated protein biogenesis factors (RPBs) act during a short but critical period of protein biogenesis. The action of RPBs starts as soon as a nascent polypeptide becomes accessible from the outside of the ribosome and ends upon termination of translation. In yeast, RPBs include the chaperones Ssb1/2 and ribosome-associated complex, signal recognition particle, nascent polypeptide-associated complex (NAC), the aminopeptidases Map1 and Map2, and the Nalpha-terminal acetyltransferase NatA. Here, we provide the first comprehensive analysis of RPB binding at the yeast ribosomal tunnel exit as a function of translational status and polypeptide sequence. We measured the ratios of RPBs to ribosomes in yeast cells and determined RPB occupation of translating and non-translating ribosomes. The combined results imply a requirement for dynamic and coordinated interactions at the tunnel exit. Exclusively, NAC was associated with the majority of ribosomes regardless of their translational status. All other RPBs occupied only ribosomal subpopulations, binding with increased apparent affinity to randomly translating ribosomes as compared with non-translating ones. Analysis of RPB interaction with homogenous ribosome populations engaged in the translation of specific nascent polypeptides revealed that the affinities of Ssb1/2, NAC, and, as expected, signal recognition particle, were influenced by the amino acid sequence of the nascent polypeptide. Complementary cross-linking data suggest that not only affinity of RPBs to the ribosome but also positioning can be influenced in a nascent polypeptide-dependent manner.