Kenneth S. Schmitz

Influence of ionic strength on the diffusion of polystyrene latex spheres, bovine serum albumin, and polynucleosomes

Quasielastic light scattering methods were used to determine apparent diffusion coefficients (Dapp) for polystyrene latex spheres, bovine serum albumin, and polynucleosomes under conditions of neutral pH and low ionic strength (0.1–50 mM). Data were collected at several time intervals (Δt) at each of several scattering angles (θ) in the range 35<θ<120 and analyzed by the asymptotic analysis method (Dapp vs T, where T=NΔt is the time window for the N+1 point autocorrelation function). These data suggest that the asymptotic limit Dapp(T → 0) is independent of θ for the three systems examined, whereas the asymptotic limit Dapp(T → ∞) appears to be proportional to the reciprocal of the total light scattering intensity. These results are interpreted in terms of ‘‘free’’ and ‘‘temporal aggregate’’ domains within the polyelectrolyte solution. The critical size, hence stability, of the temporal aggregate is proposed to result from a balance between attractive forces arising from fluctuations in the small ion and ...

Quasielastic light scattering by biopolymers. Conformation of chromatin multimers

Abstract Chicken erythrocyte chromatin was partially digested with micrococcal nuclease and separated into multimeric subunit fractions by gel permeation chromatography. The fractions were characterized by their Svedberg constant, diffusion coefficient, circular dichroism, and electrophoresis pattern of the extracted DNA. The molecular weight dependence of the sedimentation coefficient was found to be S20,w = .011 × M.554. The molecular weight dependence of rmf f o is best represented in the Kirkwood theory by either a helical superstructure or a flexible coil with attractive interactions between nucleosome units. The dimer calculations of f f o suggest that the core particles are separated by spacer regions which contribute up to ∼20% of the frictional properties of the molecule.

Quasielastic light scattering by biopolymers. Center-of-mass motion of DNA in the presence of a sinusoidal electric field

Abstract Quasielastic light scattering is now a well-established technique for rapid determination of diffusion coefficients from the spectral density of Rayleigh scattered light. Application of a constant electric field in the course of a quasielastic light scattering experiment, a technique termed electrophoretic light scattering (ELS), results in a series of peaks that are Doppler shifted by an amount proportional to the superimposed instantaneous velocity of the molecules. Electrophoretic mobilities, diffusion coefficients, and relative concentrations of each component in a polydispersed system can be determined from a single ELS experiment. A theory for polymer dynamics in the presence of an applied sinusoidal field has also been proposed. The present communication presents data in which the center-of-mass of DNA is studied in the presence of a low frequency sinusoidal field. It is shown that: (1) at very low frequencies the power spectrum is composed of several peaks Doppler shifted by an amount proportional to the driving frequency ω (i.e. fundamental and harmonic overtones); (2) the peak amplitude of the fundamental frequency shift is proportionall to 1/ω in accordance with the theory. The advantage of using a sinusoidal applied field instead of a pulsed square-wave is discussed.

Quasi-elastic light scattering studies on T7 DNA in the presence of a sinusoidal electric field

Abstract Quasi-elastic light scattering studies on T7 DNA were performed with a sinusoidal electric field applied across the sample. The resulting spectral densities exhibited several peaks whose number and magnitude depended upon the parameter z ( = μ EK cos(φ/2)/2π v ′, where μ is the apparent electrophoretic mobility. E is the magnitude of the applied electric field of frequency v ′. K is the scattering vector, and φ is the scattering angle). These data suggest two asymptotic values for the electrophoretic mobility, the high-frequency limit being associated with the center-of-mass motion of the T7 DNA and the low-frequency limit being identified with small-ion—polyion coupled motions.

Low frequency dispersion in the electrophoretic mobility of DNA and mononucleosomes

The technique of applying a sinusoidal electric field across the sample in the course of a quasielastic light scattering experiment (QELS‐SEF) is similar in design to dielectric relaxation methods except the spectral density of the scattered light is monitored instead of the electrical properties of the system. The QELS‐SEF technique was employed in a study of T7 DNA and mononucleosomes for driving frequencies of less than 100 Hz. These data suggest a dispersion in the electrophoretic mobility similar to that reported for dielectric relaxation on polynucleotides. These data are interpreted in terms of collective motions of the polyions as mediated by fluctuations in the distribution of small ions.

Conformation of Polynucleosomes in Low Ionic Strength Solution

Chromatin is a nucleohistone complex which exhibits alternating nuclease accessible and resistant regions.1–12 The repeat unit, or nucleosome, consists of approximately 200 base pairs of DNA and two each of the histones H2A, H2B, H3, and H4. The nuclease resistant region is composed of approximately 140 base pairs of DNA wrapped around the eight-histone aggregate to form a “core particle”.8,9 The remaining 60 base pairs of DNA in the nucleosome unit serve to link core particles, and hence are called “spacer DNA” or “linker DNA”. Neutron scattering studies on isolated core particles have verified the placement of DNA exterior to the protein13 and provided a structural model14–16 for the intact core particle8,9 which is in good agreement with electron microscopy studies of unstained, unfixed samples of H1 depleted core particles.17

Generation of a third-order folded structure for chromatin

Abstract A model for the initiation of the diffuse-condensed transition of chromatin induced by a change in the conformation of lysine-rich histones is proposed. Three levels of folded structures are discussed. The first-order folded structure refers to the structure of the repeat unit of chromatin, which is called the nucleosome. The nucleosome contains a nuclease resistant region in which 140 base pairs of DNA are wrapped around the surface of a histone aggregated of two copies each of the histones H2A, H2B, H3 and H4. This DNA-histone aggregate is called a core particle. The nuclease accessible region of the nucleosome is approximately 60 base pairs of DNA which link the core particle, hence the terminology “linker DNA.” The lysine-rich histones, (Hl, H5), which are more loosely bound than the core histones, are associated with the linker DNA. The second-order folded structure refers to the conformation of a polynucleosome. Based on neutron scattering and quasielastic light scattering studies the second-order folded structure is assumed to be an extended helix in solution with 5–7 nucleosome units per turn. The third-order folded structure is defined as that structure resulting from the first stage in the condensation process induced by a conformational change in the lysine-rich histones. Generation of the third-order folded structure in the proposed model is effected by an increased affinity of the lysine-rich histones for super-helical DNA in the core particles in adjacent turns of the second-order folded structure. Since the lysine-rich histones preferentially bind to A-T rich regions in DNA, the distribution of these regions would determine the third-order folded structure. The net effect of a non-random distribution of A-T rich regions as in the proposed model is the generation of a helix for the third-order folded structure. The assumption of a non-random distribution of A-T rich regions is indirectly supported by proflavine binding studies reported herein and by the existence of repetitive and non-repetitive DNA regions inferred from renaturation studies. One consequence of the proposed mechanism is that the majority of the A-T rich regions are in the interior of the third-order folded structure. Promoter sites of high A-T content would then be inaccessible to polymerases. The proposed model also suggests a role for spacer DNA in the genome. Higher order folded structures must also be present in the final state of condensed chromatin since the three orders of folded structures considered in this communication accounts for only 2% of that required in the diffuse-condensed transition.

Basic Concepts of Light Scattering

“The White Rabbit put on his spectacles. ‘Where shall I begin, please your Majesty?’ he asked. ‘Begin at the beginning,’ the King said, very gravely, ‘and go on till you come to the end: then stop.’ There was dead silence in the court, whilst the White Rabbit read out these verses:–”

Quasielastic light scattering by flexible polymers in the presence of a sinusoidal driving field: internal relaxation modes

Abstract Application of an applied electric field to a system of charged molecules superimposes a constant drift velocity on the random thermal motions of the molecules. The spectral density of light scattered from this system is frequency shifted by an amount proportional to the electrophoretic mobility of the molecule. Current theories consider only the application of a square-wave field and the effect on the center-of-mass motion of the molecule. A theory is developed in the present communication that considers the effect of a sinusoidal field on the internal motions of random coils. If the applied frequency is greater than ω D =μ EK cos(θ/2), then the center-of-mass motion remains random whereas internal modes may be “driven” by the applied field. Furthermore, the amplitude of the Doppler-shifted peak position diminishes if ωτ m > 1, where τ m is the relaxation time of the m th mode. It is possible, therefore, to obtain precise values for the number of relaxation modes present and their characteristic relaxation times.

On the possible role of small ions in the interpretation of dynamic light-scattering data

Abstract Quasi-elastic light scattering (QELS) data suggest a somewhat bizarre ionic strength dependence for the dynamics of poly(lysine). Upon decreasing the concentration of added salt, the apparent diffusion coefficient (Dapp) computed from the QELS data first increases in value and then catastrophically drops by over an order of magnitude at a certain critical salt concentration (Cc) whose value is a function of the lysine residue concentration (C′p) and not the molecular weight of the polyion. In contrast, other physical properties do not undergo such an abrupt variation in value but exhibit relatively mild changes over the same added salt range, i.e., the tracer diffusion coefficient (DTr), relative viscosity, and relative conductivity. We report in this communication QELS and fluorescent photobleaching recovery (FPR) data on poly(lysine), which corroborate the differences in the added salt behavior of Dapp and DTr previously reported in the literature. These observations are interpreted in terms of small ion effects on both the dynamics and the scattering power of the polyions. We examine the possible role of a shell of small ions about the polyion scattering unit using expressions derived by M. Kerker and co-workers [M. Kerker, J. Opt. Soc. Amer. 65, 376 (1975); M. Kerker, D. D. Cooke, and W. D. Ross, Paint Res. Inst. Proc. 47, 33 (1975); H. Chew and M. Kerker, J. Opt. Soc. Amer. 66, 445 (1976)] for the scattering power for confocal ellipsoids of different dielectric properties.