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.     

Rotational effects in quasielastic laser light scattering from T-even bacteriophage.

We have applied a theory of dynamic light scattering from large anisotropic particles, developed by Aragón and Pecora [J. Chem. Phys. 66 , 2506–2516 (1977)] to calculate the scattering expected from T-even phage models. The results indicate that the off-center rotation of the massive virus head with respect to the center of frictional resistance introduces significant rotational contributions to the light-scattering time autocorrelation function. The effect is particularly important when the fibers of the phage are extended. Reanalysis of previously published data [J. B. Welch III and V. A. Bloomfield, Biopolymers 17 , 2001–2014 (1978)], taking into account rotational corrections, confirms the equality of molecular weights of the slow- and fast-sedimenting forms of T2L bacteriophage.     

A hydrodynamic study of collagen fibrillogenesis by electric birefringence and quasielastic light scattering

Neutral soluble collagen was extracted from lathyritic rat skin under proteolysis-inhibited conditions. Purified solutions were characterized by electric birefringence and heterodyne beat quasi-elastic light-scattering techniques under conditions where the monomeric form was stable (at 4 degrees C in 0.032 M phosphate buffer at pH 7.04). Solutions were then heated and the birefringence and light scattering followed during the fibrillogenesis reaction. The monomer presents a translational diffusion coefficient of 0.85 X 10(-7) cm2/s and a rotary diffusion coefficient of 1150 +/- 50 s-1; these values are consistent with a rodlike molecular model of 220 +/- 10 nm length and 4 +/- 1 nm diameter, substantially different from electron microscopic values of 290 and 1.5 nm, respectively. We propose that at pH 7.04 and relatively high ionic strength, the collagen monomer unit must exhibit substantial deviation from a completely rigid and extended rodlike structure. During the entire lag phase in a thermally induced fibrillogenesis reaction, the relaxation times for both translational and rotational motion remain virtually unchanged. The monomer polarity is also unchanged, as shown by reverse pulse birefringence data. No intermediate size soluble aggregates, such as dimers or trimers, have been detected between monomer and very large aggregates or fibrils during the process, although early multistep assembly products (dimers, trimers) could have been seen if present. These data suggest a model for fibrillogenesis emphasizing a monomer-related nucleation event, such as internal stiffening or conformational transition, followed by a rapid continuous growth up to large fibrils.     

Conformational change of core particles studied by quasielastic light scattering

The diffusion translational coefficient D_T of core particles in monodisperse solutions has been measured by the quasielastic light scattering method in a large scale of salinities over the range 6.10⁻⁴ to 2M Na⁺ or K⁺. The observed values of D_T are independent of particle concentration in the range 0.1–2 mg/ml and do not vary with the scattering vector q corresponding to scattering angles between 40°–120°. When the salinity is progressively raised an increase of D_T from 1.9.10⁻⁷ cm²s⁻¹ to 3.2.10⁻⁷ cm²s⁻¹ was observed at about 2.10⁻³ M NaCl followed by a decrease of D_T beyond 0.6 M NaCl.The various possible causes of the changes of D_T such as interactions between particles or between particles and salt ions are discussed. We show that the single low ionic strength change is due to a conformational transition of the core particles, while the second variation of D_T accompanies the disorganization of the core particles.     

Rigidity of fibrin gels as measured by quasielastic light scattering

The strength of fibrin gels has been investigated by a recently developed laser light scattering technique for determining the shear modulus of soft gels. By this method, changes in the modulus were monitored as a function of time without perturbing the material. Fibrin gels were crosslinked with blood coagulation factor XIII. Rigidity measurements and SDS–polyacrylamide gel electrophoresis were used to correlate gel strength with the number of covalently bonded subunit chains. The modulus was found to vary linearly with the number of crosslinks until maximum rigidity was achieved.     

The osmotic response of large unilamellar vesicles studied by quasielastic light scattering

Large unilamellar vesicles of two phosphatidylcholines, one saturated (DMPC) and the other unsaturated (DOPC), prepared by the reverse-phase evaporation method were studied using the quasielastic light scattering technique. The accurate sizing obtained by this technique showed an osmotic response for the two kinds of vesicles when the salinity of the external medium was diluted. The elastic moduli of lipid vesicles bilayers in the liquid phase were then estimated according to the elasticity theory of spherical shells taking into account salt leakage data known from the literature.     

Difference in the hydration water mobility around F-actin and myosin subfragment-1 studied by quasielastic neutron scattering

Hydration water is essential for a protein to perform its biological function properly. In this study, the dynamics of hydration water around F-actin and myosin subfragment-1 (S1), which are the partner proteins playing a major role in various cellular functions related to cell motility including muscle contraction, was characterized by incoherent quasielastic neutron scattering (QENS). The QENS measurements on the D₂O- and H₂O-solution samples of F-actin and S1 provided the spectra of hydration water, from which the translational diffusion coefficient (D_T), the residence time (τ_T), and the rotational correlation time (τ_R) were evaluated. The D_T value of the hydration water of S1 was found to be much smaller than that of the hydration water of F-actin while the τ_T values were similar between S1 and F-actin. On the other hand, the τ_R values of the hydration water of S1 was found to be larger than that of the hydration water of F-actin. It was also found that the D_T and τ_R values of the hydration water of F-actin are similar to those of bulk water. These results suggest a significant difference in mobility of the hydration water between S1 and F-actin: S1 has the typical hydration water, the mobility of which is reduced compared with that of bulk water, while F-actin has the unique hydration water, the mobility of which is close to that of bulk water rather than the typical hydration water around proteins.     

A laser light scattering study of gellan gels

A study of gellan has been made using the technique of photon correlation spectroscopy. It has been confirmed that gellan gels are largely stationary at a molecular level like other polysaccharide gels and quite unlike the gels of flexible polymers such as polyacrylamide. Solution-gel transitions of deacetylated gellan in 0.025MNaCl have been studied both as a function of concentration and temperature, and the results compared with those of a parallel investigation of agarose. The interstitial spaces within gellan gels have also been studied by measuring the diffusion coefficients of dextran fractions within the gels. Since all gels are nonergodic systems, the theory of dynamic light scattering from such systems is discussed insofar as it affects the present work. It has been shown that the gellan and agarose aqueous systems are fundamentally different, in that agarose does not from a solution at very low concentrations, but splits up into macroscopic gel particles. At very low concentrations, gellan forms a solution in the presence of both gelleing and nongelling ions, the molecules of which shows little change in hydrodynamic diameter with temperature in the range 20–80°C. At higher concentrations where gels are formed, both gellan and agarose exhibit hystersis in their tempertature transitions from gel to solution and solution to gel, the solution being of large molecular aggregates. The transitions are sharp, but in both cases ther is a continous rearrangement in the structural morphology over the entire temperature range on heating, rendering the system more homogeneous prior to dissociation. In the case of gellan, however, there are two distincit phases in these structural changes—this is not true of agarose. The mean mass per unit length of the gellan fibre in the presence of 0.025M NaCl is 19 k daltons/nm at 0.7% concentration and varies with concentration to the power 0.15. The mass per unit length of the agarose fibre is much larger (ca. 110 k Daltons/nm), this difference being consistent with the difference in properties at very low concentrations. © 1994 John Wiley & Sons, Inc.     

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.     

Self-assembly of melanin studied by laser light scattering

The unknown molecular weight and chemical structure of melanin place the study of these pigments outside the range of the classical biochemical techniques; thus in this paper the problem of characterizing these heterogeneous biopolymers was approached by means of light scattering techniques, static and dynamic. The static technique allowed us to identify the macromolecular properties (MW and R(g)(2)(1/2)) of melanin extracted from sepia inksac and of two synthetic analogues: L-Dopa melanin obtained by autooxidation and by enzymatic oxidation by Tyrosinase. By dynamic light scattering (DLS), the hydrodynamic radius R(h) was measured to monitor the temporal behaviour of the polymerization and aggregation processes and R(h) variation by changing the chemical constraints of the polymerization medium, such as pH and ionic strength. The fractal dimension d of the aggregates of melanin, both natural and synthetic, in the past only recognized during the aggregation of the synthetic one by lowering the pH of the medium, was a useful parameter to further investigate and compare the structure of melanin granules of differing origins, revealing for the natural sample, a structure with clusters that are spherical, not largely hydrated and self-assembled, following a reaction limited aggregation kinetics (d=2.38).     

Dynamic light scattering study of suspensions of purple membrane

Purple membrane from Halobacterium halobium in suspensions has been studied by quasielastic light scattering. The intensity correlation functions of polarized scattered light were measured at various K2 values (K being the magnitude of the scattering vector), and the first cumulant Gamma of the field correlation function G1(tau) was obtained by a cumulant expansion method. The apparent diffusion coefficient Gamma /K2 did not increase monotonically with K2 values and showed a distinct anomaly in an intermediate range of K. A theoretical formulation of G1(tau) for a disc and an extremely oblate ellipsoidal shell of revolution (S. Fujime and K. Kubota, Biophys. Chem. 23 (1985) 1) was applied to the analysis of the spectra, and characteristic features of experimental spectra were well reproduced. It was suggested that a strong interference effect between scattered rays on Gamma /K2 should be attributed to a slight noncircular shape of the purple membrane and that a contribution to Gamma /K2 from membrane flexibility should be taken into account. This study will provide experimental evidence of the feasibility of membrane studies by dynamic light scattering.     

A rapid mixing device for quasielastic light scattering studies of reacting systems

A simple mixing device for studying fast reactions by quasielastic light scattering is described. The convection due to mixing is minimized and rapidly damped, so that light scattering measurements can be made immediately after mixing.     

The effect of histone H1 on the compaction of oligonucleosomes. A quasielastic light scattering study

The structural properties of H1-depleted oligonucleosomes are investigated by the use of quasielastic laser light scattering, thermal denaturation and circular dichroism and compared to those of H1-containing oligomers. To obtain information on the role of histone H1 in compaction of nucleosomes, translational diffusion coefficients (D) are determined for mono-to octanucleosomes over a range of ionic strength. The linear dependences of D on the number of nucleosomes show that the conformation of stripped oligomers is very extended and does not change drastically with increasing the ionic strength while the rigidness of the chain decreases due to the folding of linker DNA. The results prove that the salt-induced condensation is much smaller for H1-depleted than for H1-containing oligomers and that histone H1 is necessary for the formation of a supercoiled structure of oligonucleosomes, already present at low ionic strength.     

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.     

Motility of bovine spermatozoa studied by laser light scattering

Laser light scattering has been employed to determine the swimming speed distribution and the fraction of motile cells in samples of bovine spermatozoa. As predicted from theory, average trajectory velocities determined by laser light scattering were approximately four times the average translational speed estimated using light microscopy. The proportion of motile spermatozoa decreased with time at the same rate when samples were prepared in either HEPES or phosphate buffers. However, whereas the mean swimming velocity declined slowly in HEPES buffer, it dropped rapidly when phosphate buffer was used. Dilution (in the range 40–0.4×10⁶ spermatozoa·ml^-1) in either of these two buffers reduced the fraction of motile spermatozoa in the sample, but the mean swimming velocity of the remaining active spermatozoa was unchanged. Lowering the temperature from 37° C to 15° C reduced the mean swimming speed by a factor of 2–3 and the fraction of motile cells by a factor of 4–5.     

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.