Dynamic light scattering from solutions of microtubules.

Calf brain microtubule protein was assembled in vitro to form dilute solutions of microtubules (240 A diameter) having lengths greater than 1 micrometer. The microtubule solutions were examined by dynamic laser light scattering techniques. The angular dependence of the correlation function leads to the conclusion that the correlation function is measuring the translational diffusion constant of the particles. The length dependence of the correlation function, however, shows that the translational diffusion constant is not being measured and that the diffusion constant for the microtubules cannot be straightforwardly determined. These results suggest that a collective property of the rods is being measured by the laser light scattering. Although specific microtubule-microtubule interactions are a possible explanation for the observed results, we present arguments that suggest that the solution can be adequately modeled as a network of entangled polymers.     

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.     

Photon correlation spectroscopy and light scattering of eye lens proteins at high concentrations.

The bovine eye lens protein, alpha L crystallin, has been studied with photon correlation spectroscopy and statical light scattering in the concentration range up to 200 g/l in different solvent conditions. At higher concentration (c greater than 70 g/l) the scattering behavior is quite complicated, which results in nonexponential correlation functions. Three methods have been used for the analysis of these correlation functions, namely, cumulant analysis, sum of two exponentials analysis, and exponential sampling method. These methods resulted in very similar results. The highly concentrated solutions contain two scattering entities: the single alpha L crystallin and a rather heterogeneous population of large clusters. The statical light-scattering experiments can be interpreted in the same way and gave consistent results for the dimensions of the large scattering units. The formation of these clusters, which are strong light scatterers, is superimposed on an increasing degree of correlation between the bulk of the alpha L-crystallins, resulting in a net decrease of light scattering as a function of concentration.     

Dynamic light scattering by kappa- and lambda-carrageenan solutions

The intensity correlation functions of kappa- and lambda-carrageenan in various salt solutions and at different concentrations have been determined with the help of dynamic light scattering. From the first cumulant of these correlation functions the values of the translational diffusion coefficients D have been derived. They increase with macromolecular concentration. The extrapolated values to infinite dilution of the diffusion coefficients increase with increasing salt concentration as expected from the salt concentration dependence of the r.m.s. radii of gyration determined previously by static light scattering. The translational diffusion coefficient of lambda-carrageenan in 0.1 M NaCl is smaller than the corresponding value for the kappa species. This is consistent with the difference in contour length and linear charge density of the two samples used. No satisfactory interpretation for the concentration dependence of the diffusion coefficient seems to be possible at present. Although current theories for the macromolecular and salt concentration dependence of D, taking into account charge effects, seem to be applicable, they do not allow for a consistent interpretation of the data. No specific difference between the solution behaviour of kappa- and lambda-carrageenan has been detected.     

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.     

Dynamic light scattering study of muscle F-actin

By use of digital autocorrelation and fast Fourier methods, dynamic light-scattering studies of in vitro reconstituted muscle F-actin were made over a wide range of concentrations, 0.01-2 mg/ml F-actin. Measurements of correlation function [g1(t)]2 showed that a transition from a dilute to a semidilute regime for the Brownian motions of filaments occurred at around 0.3 mg/ml F-actin. Beyond this concentration, profiles of successively measured [g1(t)]2 showed very poor reproducibility. This resulted from the existence of very slow components, which could not be measured with a high statistical accuracy even for a measuring time of 3600 s/run. On the other hand, subtraction of these components automatically by an electronic circuit, [g-1(t)]2, or by computer processing, [g1(t)]2, resulted in a fairly good reproducibility of the profiles. The decay characteristics of [g1(t)]2 (and [g-1(t)]2) were very similar to those of [g1(t)]2 for dilute solutions. A theoretical model will be discussed which could account for the above situation. The time sequence [n(t,T)] of photoelectron counts at a sampling time T of light scattered from semidilute solutions of F-actin was stored on magnetic tapes, and both power spectra S(f) and correlation functions [g-1(t)]2 were computed by taking the ensemble average over many short records with duration 1024T. Since both S(f) and [g-1(t)]2 lacked frequency components lower than 1/(2048T) Hz, their profiles were highly reproducible. An analysis of S(f) confirmed our earlier results which had shown an apparent contradiction to later results by a correlation method. A comparison of S(f) and [g-1(t)]2 based on the same [n(t,T)] clarified the reasons why the bandwidth gamma of S(f) largely differed from the bandwidth gamma of [g1(t)]2 and [g-1(t)]2. The temperature dependence of gamma suggested that F-actin would be flexible and that the flexibility parameter would change with temperature.     

Photon and fluorescence correlation spectroscopy and light scattering of eye-lens proteins at moderate concentrations

The bovine eye-lens protein, alpha L-crystallin, has been studied with photon correlation spectroscopy to obtain the mutual diffusion coefficient, Dm, with fluorescence correlation spectroscopy to determine the tracer diffusion coefficient, DT, and with light scattering to get the isothermal osmotic compressibility (delta pi/delta c) P,T. The concentration dependence of Dm, DT, and (delta pi/delta c) P,T up to a volume fraction phi of the protein of 2.5 x 10(-2) has been interpreted on the basis of four different interaction potentials: (a) an extended hard-sphere potential; (b) a shielded Coulomb potential; (c) a shielded Coulomb interaction where the effect of counterions is included; (d) a simple mixed potential. The three parameters Dm, DT, and (delta pi/delta c) P,T have also been combined in the generalized Stokes-Einstein equation, Dm = [(delta pi/delta c)P,T . (1--phi) . (DT)]/(kappa B . T). Our results indicate that, in the case that photon correlation spectroscopy gives the mutual diffusion coefficient Dm, the applicability of the Stokes-Einstein equation can be questioned; or that, when one assumes the Stokes-Einstein equation to be valid, there is significant discrepancy between the result of photon correlation spectroscopy and Dm.     

Dynamic light scattering study of calmodulin-target peptide complexes

Dynamic light scattering (DLS) has been used to assess the influence of eleven different synthetic peptides, comprising the calmodulin (CaM)-binding domains of various CaM-binding proteins, on the structure of apo-CaM (calcium-free) and Ca(2+)-CaM. Peptides that bind CaM in a 1:1 and 2:1 peptide-to-protein ratio were studied, as were solutions of CaM bound simultaneously to two different peptides. DLS was also used to investigate the effect of Ca(2+) on the N- and C-terminal CaM fragments TR1C and TR2C, and to determine whether the two lobes of CaM interact in solution. The results obtained in this study were comparable to similar solution studies performed for some of these peptides using small-angle x-ray scattering. The addition of Ca(2+) to apo-CaM increased the hydrodynamic radius from 2.5 to 3.0 nm. The peptides studied induced a collapse of the elongated Ca(2+)-CaM structure to a more globular form, decreasing its hydrodynamic radius by an average of 25%. None of the peptides had an effect on the conformation of apo-CaM, indicating that either most of the peptides did not interact with apo-CaM, or if bound, they did not cause a large conformational change. The hydrodynamic radii of TR1C and TR2C CaM fragments were not significantly affected by the addition of Ca(2+). The addition of a target peptide and Ca(2+) to the two fragments of CaM, suggest that a globular complex is forming, as has been seen in nuclear magnetic resonance solution studies. This work demonstrates that dynamic light scattering is an inexpensive and efficient technique for assessing large-scale conformational changes that take place in calmodulin and related proteins upon binding of Ca(2+) ions and peptides, and provides a qualitative picture of how this occurs. This work also illustrates that DLS provides a rapid screening method for identifying new CaM targets.     

Dynamic light scattering study of calcium-induced fusion in phospholipid vesicles.

Acidic sonicated phospholipid vesicles can undergo dramatic morphological changes due to fusion in the presence of divalent metal ions. For example, small spherical phosphatidylserine vesicles can form scroll-like cylinders which precipitate in the presence of Ca2+ above a threshold concentration. Subsequent addition of EDTA will yield large, unilamellar vesicles. These events have previously been established through the combined use of differential scanning calorimetry and freeze-fracture electron microscopy. We have applied the technique of dynamic light scattering to follow these fusion events rapidly, accurately, and non-perturbatively as they occur in solution at calcium concentrations slightly below threshold for precipitation.     

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.     

Quasi-elastic laser light scattering from solutions and gels of hemoglobin S.

Quasi-elastic light scattering has been used to examine solutions and gels of deoxyhemoglobin S. The autocorrelation function is found to decay with a characteristic exponential relaxation which can be ascribed to the diffusion of monomer (64,000 molecular weight) hemoglobin S molecules. In the absence of polymers, the relaxation time is in good agreement with previous measurements of the diffusion coefficient for solutions of normal human hemoglobin. In the presence of the polymer phase, a large (greater than 200-fold) increase in the scattered intensity is observed but no contribution to the decay of the autocorrelation function from the motion of the aligned polymer phase can be detected. Heterodyning between the time-independent scattering amplitude from the polymers and the time-dependent scattering of the diffusing monomers results in a twofold increase in the relaxation time arising from monomer diffusion.     

Dynamic light scattering of biopolymers and biocolloids.

Widespread applications of dynamic light scattering techniques to the study of macromolecular Brownian motion have yielded not only a valuable store of factual information concerning solution conformations and conformational changes, but have also provided an important window through which to view the dynamics of internal modes of motion. These techniques have coincided with a resurgence of interest in the solution physical chemistry of macromolecules, including hydrodynamic properties, and the profound effect of intermolecular interactions on both the disposition and dynamics of macromolecules in solution.     

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.     

Dynamic light scattering study of calcium-induced fusion in phospholipid vesicles

Acidic sonicated phospholipid vesicles can undergo dramatic morphological changes due to fusion in the presence of divalent metal ions. For example, small spherical phosphatidylserine vesicles can form scroll-like cylinders which precipitate in the presence of Ca²⁺ above a threshold concentration. Subsequent addition of EDTA will yield large, unilamellar vesicles. These events have previously been established through the combined use of differential scanning calorimetry and freeze-fracture electron microscopy. We have applied the technique of dynamic light scattering to follow these fusion events rapidly, accurately, and non-perturbatively as they occur in solution at calcium concentrations slightly below threshold for precipitation.     

Dynamic light scattering investigations of human erythrocyte spectrin.

Dynamic light scattering measurements were performed on spectrin from human erythrocytes in 25 mM Tris buffer at pH 7.6 with 100 mM NaCl and 5 mM EDTA. Measurements were made on spectrin solutions prepared as dimers and tetramers over the temperature range from 23 to 41 degrees C, as a function of the square of the scattering vector (K2) over the range of 0.7 x 10(10) cm-2 less than or equal to K1 less than or equal to 20 x 10(10) cm-2. Analysis of the autocorrelation functions collected for these solutions revealed the presence of two predominant motional components over the entire range of K2. Plots of the diffusion coefficients (D20) of these components, with viscosity and temperature corrected to water at 20 degrees C, as a function of K2 indicated three rather distinct regions, flat regions at low and high K2 joined by a sloping intermediate region. At small K2 (less than or equal to 4 x 10(10) cm-2) the D20 values were (7.3 +/- 2.0) x 10(-8) cm2/s for the slow component and (20.3 +/- 2.0) x 10(-8) cm2/s for the fast component. At large K2 (greater than or equal to 10 x 10(10) cm-2) the values increased to (13.0 +/- 2.0) x 10(-8) cm2/s for the slow component and (39.4 +/- 2.0) x 10(-8) cm2/s for the fast component. In the intermediate K2 region, D20 is a linear function of K2 and appears as a transition between the low and high K2 regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic light scattering studies of ribonuclease

Dynamic light scattering has been used to measure the translational diffusion coefficients of bovine pancreatic ribonuclease A as functions of temperature and concentration in the presence of 1 M Guanidine-HCl. Data was collected throughout a temperature range including the folding-unfolding transitions. Evidence of a pretransition "swelling" of the protein was observed. Entropy and enthalpy changes upon unfolding were obtained using a two-state model.     

Internal dynamics of tRNA(Phe) studied by depolarized dynamic light scattering

The collective internal dynamics of transfer RNA(Phe) from brewer's yeast in solution was studied by depolarized dynamic light scattering (DDLS). Within the melting region of tRNA the depolarized spectra consist of two Lorentzian, where the narrow (slow) component describes the overall rotation of the macromolecule. The broad component is attributed to the collective reorientation of the bases within the biopolymer. At high temperature only this relaxation process is observed in the spectrum. The viscosity dependence of the collective internal relaxation process is described by the Stokes-Einstein-Debye equation for rotational diffusion. Estimates of the internal orientational pair correlation factor from the integral depolarized intensities of tRNA(Phe) solutions indicates that the observed dynamics correspond to the collective reorientation of approximately 5 bases. A comparison of the results presented with DDLS studies on the aggregation of the mononucleotide guanosine-5'-monophosphate confirms this result. For a further characterization of the relaxation process we studied the effect of hydrostatic pressure (1-1000 bar) on the depolarized spectra of tRNA. While other spectroscopic methods like nmr, fluorescence polarization anisotropy decay, or ESR give information about the very local motion of a single base within the DNA or RNA, this study shows that by DDLS one can characterize collective internal motions of macromolecules.