Measurement of particle sizes by elastic and quasi-elastic light scattering.

A method of measuring an average particle radius in a highly polydisperse dispersion using the wavelength dependence of turbidity is described. For particles which are no larger than 0.3 of the wavelength of light used, a polynomial representation of the scattering cross-section can be used. For larger particles, more extensive numerical calculations are required. The use of the method is illustrated by determining the average particle radius of casein micelles by both elastic and quasi-elastic light scattering techniques. A polydisperse homogeneous sphere model is found to be a reasonably accurate representation of casein micelles. Several modifications of the model which would improve the agreement between the two techniques are mentioned briefly.     

A study of the dynamic properties of actomyosin systems by quasi-elastic light scattering.

A laser light source and a digital autocorrelator were employed in the study of the molecular dyanmics of acto-heavy meromyosin during the splitting of ATP. Low protein concentrations were used, so that molecular and not gel properties were evident. The addition of Mg2+ to acto-heavy meromyosin solutions in the presence of ATP caused a marked widening of the spectrum at high scattering angles. No such change was observed when chemically inactivated heavy meromyosin was used when actin was cross-linked or when the proteins were in a high ionic strength solution. The data can be interpreted in terms of pronounced change in flexibility of acto-heavy meromyosin induced by active mechanochemical coupling.     

Configurational and dynamic properties of different length superhelical DNAs measured by dynamic light scattering

The solution conformation and internal motions of five superhelical DNAs between 2100 and 10200 base-pairs in length have been characterized by dynamic light scattering (DLS). Variations in the diffusion coefficients and rotational relaxation times with molecular weight are both indicative of an anisotropic extended structure of these DNAs; we therefore conclude that under our conditions the interwound superhelical structure prevails. The internal dynamics can be described by a superposition of rotational diffusion and internal relaxation. The latter process is characterized by the internal diffusion of persistence length size segments within the DNA chain and faster bending motions within these segments.     

Vesicle sizing: Number distributions by dynamic light scattering

A procedure is described which optimizes nonnegative least squares and exponential sampling fitting methods for analysis of dynamic light scattering (DLS) data from aqueous suspensions of vesicle/liposome systems. This approach utilizes a Rayleigh-Gans-Debye form factor for a coated sphere and yields number distributions which can be compared directly to distributions obtained by freeze-fracture electron microscopy (EM). Excellent agreement between the DLS and EM results are obtained for vesicle size distributions in the 100-200-nm range.     

A study of the dynamic properties of actomyosin systems by quasi-elastic light scattering

A laser light source and a digital autocorrelator were employed in the study of the molecular dynamics of acto-heavy meromyosin during the splitting of ATP. Low protein concentrations were used, so that molecular and not gel properties were evident. The addition of Mg²⁺ to acto-heavy meromyosin solutions in the presence of ATP caused a marked widening of the spectrum at high scattering angles. No such change was observed when chemically inactivated heavy meromyosin was used, when actin was cross-linked or when the proteins were in a high ionic strength solution. The data can be interpreted in terms of pronounced change in flexibility of acto-heavy meromyosin induced by active mechanochemical coupling.     

Characterization of precrystallization aggregation of canavalin by dynamic light scattering.

The aggregation processes leading to crystallization and precipitation of canavalin have been investigated by dynamic light scattering (DLS) in photon correlation spectroscopy (PCS) mode. The sizes of aggregates formed under various conditions of pH, salt concentration, and protein concentrations were deduced from the correlation functions generated by the fluctuating intensity of light scattered by the solutions of the protein. Results obtained indicate that the barrier to crystallization of canavalin is the formation of the trimer, a species that has been characterized by x-ray crystallographic studies (McPherson, A. 1980. J. Biol. Chem. 255:10472-10480). The dimensions of the trimer in solution are in good agreement with those obtained both from the crystal (McPherson, A. 1980. J. Biol. Chem. 255:10472-10480) and from a low angle x-ray scattering study in solution (Plietz, P., P. Damaschun, J. J. Müller, and B. Schlener. 1983. FEBS [Fed. Eur. Biochem. Soc.] Lett. 162:43-46). Furthermore, under conditions known to lead to the formation of rhombohedral crystals of canavalin, a limiting size is reached at high concentrations of canavalin. The size measured corresponds to an aggregate of trimers making a unit rhombohedral cell consistent with x-ray crystallographic data (McPherson, A. 1980. J. Biol. Chem. 255:10472-10480). Presumably, such aggregates are the nuclei from which crystal growth proceeds. The present study was undertaken primarily to test the potential of DLS (PCS) as a tool for rapid, routine screening to determine the ultimate fate of protein solutions (i.e., crystallization or amorphous precipitation) at an early stage, therefore eliminating the need for long-term visual observation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution properties of starch nanoparticles in water and DMSO as studied by dynamic light scattering

Solution properties of starch nanoparticles dispersed in DMSO and in water were studied using dynamic light scattering. The particle size distribution had two main peaks in both solvents at all scattering angles studied. They were at around 40 and 300 nm, ascribed to isolated starch nanoparticles and their aggregates, respectively. From the excess scattering intensity by the 40-nm particles, the molecular weight of the nanoparticle was estimated as 2.2–2.6×10⁶ g/mol. When the concentration was increased, another peak appeared at around 1 μm. Raising the temperature from 25 to 65 °C did not change the distribution, indicating a purely entropic process in dynamic equilibrium of the aggregation. In DMSO, an oscillatory behavior was observed in the autocorrelation function at high temperatures.     

Gelatin-alpha olefin sulfonate interactions studied by dynamic light scattering

Dynamic light scattering (DLS) measurements were performed to study the binding of anionic surfactant alpha olefin sulfonate (AOS) to gelatin chains at various NaCl concentrations at 30 degrees C in aqueous sodium phosphate buffer (pH = 6.8) solutions. The surfactant concentration was varied from 0 to 80 mM and the NaCl concentrations chosen were 0.025, 0.05, and 0.1 M. AOS exhibited electrostatic binding to the positively charged sites of the polypeptide chain resulting in considerable reduction in its hydrodynamic radius up to critical micellar concentration (cmc = 8 mM for no salt, 0.01 and 0.025 M, and 5 mM for 0.05 M and 2 mM for 0.1 M solutions). The correlation function revealed the presence of two types of structures above cmc; namely the micelles of AOS and gelatin-AOS micelle complexes. The micellar radii (Rm), the effective gelatin-surfactant complex radii (Rc), have been determined as a function of salt concentration. No critical aggregation concentration (cac) was observed. The inter-gelatin-surfactant complex (kD1) and inter-micellar interactions (kD2), were determined by fitting the concentration dependence of Rm and Rc to a virial expansion in reduced concentration (c - cmc), which are compared. While kD1 showed strong ionic strength dependence, kD2 remained invariant of the same. The protein to surfactant binding ratio was found to be smaller than normal. Results have been discussed within the framework of the necklace-bead model of polymer-surfactant interactions.     

Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy.

We present a new approach to probing single-particle dynamics that uses dynamic light scattering from a localized region. By scattering a focused laser beam from a micron-size particle, we measure its spatial fluctuations via the temporal autocorrelation of the scattered intensity. We demonstrate the applicability of this approach by measuring the three-dimensional force constants of a single bead and a pair of beads trapped by laser tweezers. The scattering equations that relate the scattered intensity autocorrelation to the particle position correlation function are derived. This technique has potential applications for measurement of biomolecular force constants and probing viscoelastic properties of complex media.     

Solution dimensions of the gramicidin dimer by dynamic light scattering

Gramicidin is thought to form dimeric helical rods in alcohol solutions. In addition, there is evidence that the rod dimensions change upon addition of potassium ions. The present work reports values for the translational and rotational diffusion coefficients of gramicidin in methanol and 95% ethanol and in these same solvents with added KSCN. Solution dimensions are calculated from the diffusion coefficients. The results suggest that gramicidin exists primarily as dimers in these solutions and that the gramicidin rod does indeed become shorter upon addition of potassium ion. These results are consistent with those obtained from X-ray studies on single crystals grown from alcohol solutions.     

Self-assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy

Dynamic light scattering (DLS) analysis together with atomic force microscopy (AFM) imaging was applied to investigate the supramolecular self-assembly properties of a series of recombinant amelogenins. The overall objective was to ascertain the contribution of certain structural motifs in amelogenin to protein-protein interactions during the self-assembly process. Mouse amelogenins lacking either amino- or carboxy-terminal domains believed to be involved in self-assembly and amelogenins having single or double amino acid mutations identical to those found in cases of amelogenesis imperfecta were analyzed. The polyhistidine-containingfull-length recombinant amelogenin protein [rp(H)M180] generated nanospheres with monodisperse size distribution (hydrodynamic radius of 20.7 +/- 2.9 nm estimated from DLS and 16.1 +/- 3.4 nm estimated from AFM images), comparable to nanospheres formed by full-length amelogenin rM179 without the polyhistidine domain, indicating that this histidine modification did not interfere with the self-assembly process. Deletion of the N-terminal self-assembly domain from amelogenin and their substitution by a FLAG epitope ("A"-domain deletion) resulted in the formation of assemblies with a heterogeneous size distribution with the hydrodynamic radii of particles ranging from 3 to 38 nm. A time-dependent dynamic light scattering analysis of amelogenin molecules lacking amino acids 157 through 173 and containing a hemagglutinin epitope ("B"-domain deletion) resulted in the formation of particles (21.5 +/- 6.8 nm) that fused to form larger particles of 49.3 +/- 4.3 nm within an hour. Single and double point mutations in the N-terminal region resulted in the formation of larger and more heterogeneous nanospheres. The above data suggest that while the N-terminal A-domain is involved in the molecular interactions for the formation of nanospheres, the carboxy-terminal B-domain contributes to the stability and homogeneity of the nanospheres, preventing their fusion to larger assemblies. These in vitro findings support the notion that the proteolytic cleavage of amelogenin at amino- and carboxy-terminii occurring during enamel formation influences amelogenin to amelogenin interactions during self-assembly and hence alters the structural organization of the developing enamel extracellular matrix, thus affecting enamel biomineralization.     

Rotational relaxation of macromolecules determined by dynamic light scattering. I. Tobacco mosaic virus

The intensity autocorrelation function for the depolarized component of forward-scattered light from a solution of large polymeric molecules is derived in terms of the correlation function for the amplitudes of the Y₂,±₁(θ,?) fluctuations in the anglar distribution of segments in the solution without any assumptions regarding the statistical properties of the scatterad light field. Effects arising from the use of polychromatic incident light and from the mixing of the scattered and polychromatic incident light beams are examined in detail. Apparatus for observing the depolarized forward-scattered light, digitizing and storing the fluctuating phototube current at rates from 10 to 540,000 times per second, and computing the correlation functions directly in the time-domain is described herein. Correlation functions were obtained for 0.05 mg/ml solution of tobacco mosaic virus at pH 9.1 and also at pH 6. The degree of association of the virus appears to be independent of pH, and the monomer relaxation times (corrected to 25°C) extracted from the data by a least-squares procedure lie in the range 0.44–0.49 msec, also independent of pH. The absence of faster component in the correlation function between 6 μsec and 0.5 msec is used in conjunction with thermal fluctuation theory to infer a lower limit for the effective Young's modulaus of the rod, E ≤ 2.5 × 10⁷ dynes/cm².     

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.     

Pressure assisted stabilization of biocatalysts at elevated temperatures: Characterization by dynamic light scattering

The effect of pressure, at elevated temperatures, is reported on the activity and stability of a thermophilic endo‐β‐glucanase from the filamentous fungus Talaromyces emersonii. The production of reduced sugars after treatment at different temperatures and pressures is used as a measure of the activity and stability of the enzyme. The activity of the enzyme is maintained to higher temperatures with increasing pressure. For example, the relative activity of endo‐β‐glucanase decreases to 30% after 4 h at 75°C and 1 bar, whereas it is preserved at 100% after 6 h at 75°C and 230 bar. High‐pressure dynamic light scattering is used to characterize the hydrodynamic radius of the enzyme as a function of pressure, temperature, and time. At higher temperature the hydrodynamic radius increases with time, whereas increasing pressure suppresses this effect. Changes in the hydrodynamic radius are correlated with the activity measurements obtained at elevated pressures, since the changes in the hydrodynamic radius indicate structural changes of the enzyme, which cause the deactivation. Biotechnol. Bioeng. 2013; 110: 1674–1680. © 2012 Wiley Periodicals, Inc.     

The onset of amelogenin nanosphere aggregation studied by small-angle X-ray scattering and dynamic light scattering

Proteins with predominantly hydrophobic character called amelogenins play a key role in the formation of the highly organized enamel tissue by forming nanospheres that interact with hydroxyapatite crystals. In the present investigation, we have studied the temperature and pH-dependent self-assembly of two recombinant mouse amelogenins, rM179 and rM166, the latter being an engineered version of the protein that lacks a 13 amino acid hydrophilic C-terminus. It has been postulated that this hydrophilic domain plays an important role in controlling the self-assembly behavior of rM179. By small-angle X-ray and neutron scattering, as well as by dynamic light scattering, we observed the onset of an aggregation of the rM179 protein nanospheres at pH 8. This behavior of the full-length recombinant protein is best explained by a core-shell model for the nanospheres, where hydrophilic and negatively charged side chains prevent the agglomeration of hydrophobic cores of the protein nanospheres at lower temperatures, while clusters consisting of several nanospheres start to form at elevated temperatures. In contrast, while capable of forming nanospheres, rM166 shows a very different aggregation behavior resulting in the formation of larger precipitates just above room temperature. These results, together with recent observations that rM179, unlike rM166, can regulate mineral organization in vitro, suggest that the aggregation of nanospheres of the full-length amelogenin rM179 is an important step in the self-assembly of the enamel matrix.