Low-angle light-scattering studies on alkali- and heat-denatured DNA

The molecular weight of native DNA is shown to decrease by at least a factor of two on denaturation by heat or alkali. This result is obtained only if low-angle (<30°) light-scattering measurements are used. High-angle measurements (>30°) do not reveal a decrease in molecular weight on denaturation due to the incorrect value for native DNA. The dn/dc value for both native and denatured DNA is 0.166 ml/g ± 0.003 ml/g. Methods are described for the clarification of native and denatured DNA solutions for light scattering by the use of membrane filters.     

Quasielastic light-scattering study of polyadenylic acid solutions. II

Quasielastic light scattering is used to study saline solutions of polyadenylic acid with varying polymer concentrations and molecular masses. These experiments clearly show the existence of two relaxation times. For dilute solutions, when the chains are mutually independent, the fast mode is due to the free diffusion of the polymer chains. For concentrations above the overlap concentration C*, the fast mode is due to the propagation of collective excitations of the pseudolattice of polymer chains. The slow modes are observed when the polymer concentration is in the vicinity of the overlap concentration C*. A series of experiments shows that both their relaxation time and amplitude depend only on the polymer concentration and not on the polymer molecular mass. This result rules out any previous explanation based on individual chain motion. Furthermore, since the amplitudes depend on the time elapsed from the preparation of the solution, the slow modes are due to the diffusion of concentration inhomogeneities in the pseudolattice.     

Development of a charged-coupled device-based light-scattering instrument for the detection of C-reactive protein using particle-enhanced immunoassay

A novel light-scattering instrument has been developed for rapid detection of immunoreactions in test latex particle-enhanced immunoassays. The detector consists of a flat-field grating and a charge-coupled device mounted on a rotating platform, and the detector measures a continuous spectrum from 350 nm to 735 nm at 440 polar angles with a resolution of 0.5 degrees. Optimal detection for rates of immunoreaction were determined by intensity of scattered light at different angles. Instrumental precisions were all shown to fall within 5% of the target relative standard deviation limits. The accuracy of the instrument was confirmed using monodispersed latex particles of known size and shape. The initial results showed the possibility of a sensitive and accurate detection of C-reactive protein throughout the range of clinical interest, thus demonstrating a significant potential for biomedical applications.     

Neutron and light-scattering studies of DNA gyrase and its complex with DNA

The solution structure of Escherichia coli DNA gyrase, an enzyme that catalyzes the ATP-dependent supercoiling of DNA, has been characterized by small-angle neutron scattering (SANS) and dynamic light-scattering (DLS). The enzyme and its complex with a 172 base-pair fragment of duplex DNA, in H2O or 2H2O solvent, were studied by contrast variation and the measurement of hydrodynamic parameters as a function of scattering angle. The complex was also measured in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP), a non-hydrolyzable ATP analog that is known to support limited supercoiling. The values of the radius of gyration, Rg = 67 A, from SANS and the hydrodynamic radius, Rh = 64 A, from DLS predict a larger than expected volume for the enzyme, supporting the notion of channels or cavities within the molecule. In addition, several classes of models were rejected based on SANS data obtained in 2H2O at larger scattering angles. The best fit to both the SANS and DLS data is obtained for oblate, inhomogeneous particles approximately 175 A wide and 52 A thick. Such particles provide a large surface area for DNA interaction. Both Rg and Rh values change very little upon addition of DNA, suggesting that DNA binds in a manner that does not significantly change the shape of the protein. No appreciable change in structure is found with the addition of ADPNP. However, the higher-angle SANS data indicate a slight rearrangement of the enzyme in the presence of nucleotide.     

Interactions of lysozyme in concentrated electrolyte solutions from dynamic light-scattering measurements

The diffusion of hen egg-white lysozyme has been studied by dynamic light scattering in aqueous solutions of ammonium sulfate as a function of protein concentration to 30 g/liter. Experiments were conducted under the following conditions: pH 4-7 and ionic strength 0.05-5.0 M. Diffusivity data for ionic strengths up to 0.5 M were interpreted in the context of a two-body interaction model for monomers. From this analysis, two potential-of-mean-force parameters, the effective monomer charge, and the Hamaker constant were obtained. At higher ionic strength, the data were analyzed using a model that describes the diffusion coefficient of a polydisperse system of interacting protein aggregates in terms of an isodesmic, indefinite aggregation equilibrium constant. Data analysis incorporated multicomponent virial and hydrodynamic effects. The resulting equilibrium constants indicate that lysozyme does not aggregate significantly as ionic strength increases, even at salt concentrations near the point of salting-out precipitation.     

Dynamic light-scattering studies of DNA. I. The coupling of internal modes with anisotropic translational diffusion in congested solutions

A model for the coupling between internal modes, or molecular rotation, and anisotropic translational diffusion in congested solutions is proposed to account for the anomalously slow component that has appeared ubiquitously in reported autocorrelation functions of Rayleigh scattered light from solutions of DNA's with molecular weights greater than about 10⁷. The predicted existence of an anomalously slow mode in addition to a faster “normal” mode, as well as the predicted relative amplitudes of both fast and slow components, are qualitatively in agreement with the observations. For sufficiently long-wavelength fluctuations all of the amplitude appears in the slower mode, which then exhibits an appropriately averaged translational diffusion coefficient. In support of the model it is shown in the Appendix that nonideal central interactions between macromolecules are by themselves insufficient to generate isolated internal mode relaxation terms in the autocorrelation function, unless translational ordering of the macromolecules extends over the illuminated observation region.     

Structural studies of the solutions of anionic polysaccharides. IV. Study of pectin solutions by light-scattering

Aqueous solutions of pectins, with a degree of esterification (DE) varying from 0 to 95%, have been studied by light-scattering. A set of samples of similar molecular sizes were prepared by methylation of sodium pectate with diazomethane. The solutions were subjected to ultracentrifugation to ensure the removal of small amounts of gel fraction. The second virial coefficient of pectin was positive and constant for DE values varying between 43 and 95%, but increased by a factor of three when the DE was reduced to zero. This increase is at least partly due to coulombic electrostatic interactions. All of the pectins investigated behaved as semi-rigid-chain polymers. The chain flexibility was a maximum in the DE range 43 to 58%.In pectin solutions with a DE higher than 58% attractive forces exist between pectin macromolecules due to the presence of ester groups. It is expected that the interactions between ester groups contribute to both the chain rigidity and the gel-forming ability of pectins.     

One- and two-soliton solutions for DNA

One- and two-soliton solutions of the nonlinear differential equation imitating rotational oscillations of DNA bases were found. It was shown that the equation is analogous to the simple sine-Gordon equation, which is solved by the inverse scattering method using the L-A pair. One- and two-soliton solutions found by the method were presented in terms of DNA parameters. The profiles of nonlinear one-soliton waves were calculated for two cases: for a homogeneous double polynucleotide chain consisting of A-T base pairs and for a homogeneous double polynucleotide chain consisting of G-C base pairs.     

Interactions of lysozyme in guanidinium chloride solutions from static and dynamic light-scattering measurements

The interactions of partially unfolded proteins provide insight into protein folding and protein aggregation. In this work, we studied partially unfolded hen egg lysozyme interactions in solutions containing up to 7 M guanidinium chloride (GdnHCl). The osmotic second virial coefficient (B(22)) of lysozyme was measured using static light scattering in GdnHCl aqueous solutions at 20 degrees C and pH 4.5. B(22) is positive in all solutions, indicating repulsive protein-protein interactions. At low GdnHCl concentrations, B(22) decreases with rising ionic strength: in the absence of GdnHCl, B(22) is 1.1 x 10(-3) mLmol/g(2), decreasing to 3.0 x 10(-5) mLmol/g(2) in the presence of 1 M GdnHCl. Lysozyme unfolds in solutions at GdnHCl concentrations higher than 3 M. Under such conditions, B(22) increases with ionic strength, reaching 8.0 x 10(-4) mLmol/g(2) at 6.5 M GdnHCl. Protein-protein hydrodynamic interactions were evaluated from concentration-dependent diffusivity measurements, obtained from dynamic light scattering. At moderate GdnHCl concentrations, lysozyme interparticle interactions are least repulsive and hydrodynamic interactions are least attractive. The lysozyme hydrodynamic radius was calculated from infinite-dilution diffusivity and did not change significantly during protein unfolding. Our results contribute toward better understanding of protein interactions of partially unfolded states in the presence of a denaturant; they may be helpful for the design of protein refolding processes that avoid protein aggregation.     

A label-free light-scattering method to resolve assembly and disassembly of DNA nanostructures

DNA self-assembly, and in particular DNA origami, has evolved into a reliable workhorse for organizing organic and inorganic materials with nanometer precision and with exactly controlled stoichiometry. To ensure the intended performance of a given DNA structure, it is beneficial to determine its folding temperature, which in turn yields the best possible assembly of all DNA strands. Here, we show that temperature-controlled sample holders and standard fluorescence spectrometers or dynamic light-scattering setups in a static light-scattering configuration allow for monitoring the assembly progress in real time. With this robust label-free technique, we determine the folding and melting temperatures of a set of different DNA origami structures without the need for more tedious protocols. In addition, we use the method to follow digestion of DNA structures in the presence of DNase I and find strikingly different resistances toward enzymatic degradation depending on the structural design of the DNA object.     

An automated instrument for the performance of enzymatic DNA sequencing reactions

An instrument has been developed for the automation of enzymatic DNA sequencing reactions. Up to 96 DNA templates contained in a microtiter plate can be processed for either radioactive or fluorescence-based sequence analysis in a three-hour period. The quality of the resultant data is comparable to that obtained manually. The system is simple, flexible and is readily adapted to the use of new polymerases or modified experimental protocols.