Desmin filaments studied by quasi-elastic light scattering.

We studied polymers of desmin, a muscle-specific type III intermediate filament protein, using quasi-elastic light scattering. Desmin was purified from chicken gizzard. Polymerization was induced either by 2 mM MgCl(2) or 150 mM NaCl. The polymer solutions were in the semidilute regime. We concluded that the persistence length of the filaments is between 0.1 and 1 microm. In all cases, we found a hydrodynamic diameter of desmin filaments of 16-18 nm. The filament dynamics exhibits a characteristic frequency in the sense that correlation functions measured on one sample but at different scattering vectors collapse onto a single master curve when time is normalized by the experimentally determined initial decay rate.     

Suppression of active cross-bridge motions of isolated thick myofilaments in suspension by phenylmethylsulfonyl fluoride

Studies of photoelectron count autocorrelation function of light scattered from suspensions of thick filaments of Limulus telson muscle and scallop striated adductor muscle reveal that Ca2+ can activate cross-bridge motions of these isolated filaments. By treating suspensions of activated filaments with phenylmethylsulfonyl fluoride (PMSF), we can suppress active cross-bridge motions but not affect the Ca2+-dependent ATPase activity.     

Myosin thick filaments from adult rabbit skeletal muscles

Myosin subfragment 1 (S1) forms dimers in the presence of Mg(2+) or MgADP or MgATP. The entire myosin molecule forms head-head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95 degrees. Assuming that the length of the globular part of S1 is approximately 12 nm and that the S1/S2 joint (lever arm approximately 7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of approximately 18 nm, close to the approximately 16-20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament-filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature ( approximately 22 degrees C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg(2+) or 0.5 mM MgATP and at low temperature ( approximately 0 degrees C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature ( approximately 0 degrees C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament-filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).     

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.     

Casein micelle size from elastic and quasi-elastic light scattering measurements.

The average molecular weight, particle radius and size distribution of particles in skim milk from eight cows in mid-lactation have been measured by means of elastic and quasi-elastic light scattering techniques. The properties of sub-micellar casein particles in the milk of each cow were also studied. Particular attention has been given to the effects of particle size heterogeneity in the interpretation of results. The weight average molecular weight of the particles from different cows varied from 2.6-10(8) to 15-10(8) and the corresponding average particle radius varied between 90 and 130 nm. An unusual feature of these particles is their high water content, which was found to vary from 2.4 to 6.4 ml/g with a positive correlation between average particle density and average particle mass. Variations in particle water content can be most readily understood in terms of a gel-like casein micelle.     

Determining molecular weight distributions of antigen-antibody complexes by quasi-elastic light scattering.

Physiological properties of soluble antigen-antibody (Ag-Ab) complexes depend in part on the size of the complexes. In previous work, the size distribution and structure of model Ag-Ab complexes were determined by electron microscopy. In this study, we used constrained regularization analysis of quasi-elastic light scattering data to estimate molecular weight distributions of model Ag-Ab complexes. A conformational model was necessary to determine appropriate correlations between molecular weight and diffusion coefficient, and to estimate particle structure factors. Porod-Kratky theory proved to be an adequate conformational model for these purposes. The molecular weight distributions determined by constrained regularization compared favorably with distributions obtained either by electron microscopy or by thermodynamic modeling.     

Histone-induced conformational changes in DNA as probed by quasi-elastic light scattering.

Quasi-elastic light scattering as measured by intensity fluctuation (self-beat) spectroscopy in the time domain can be profitably used to follow both the translational diffusion D and the dominant internal flexing mode τ_int of DNA and its complexes with various histones in aqueous salt solutions. Without histones, DNA is found to have D = 1.6 × 10^?8 cm²/sec and τ_int ? 5 × 10^?4 sec in 0.8 M NaCl, 2 M urea at 20°C. Total histone as well as fraction F2A induce supercoiling (D = 2.6 × 10^?8 cm²/sec, τ_int ? 2.8 × 10^?4 sec) whereas fraction F1 induces uncoiling (D = 1.0 × 10^?8 cm²/sec, τ_int ? 9.4 × 10^?4 sec). Upon increasing the salt concentration to 1.5 M the DNA–histone complex dissociates (D = 1.8 × 10^?8 cm²/sec). Upon decreasing the salt concentration to far below 0.8 M, the DNA–histone complex eventually precipitates as a chromatin gel.     

Properties of pancreatic zymogen granules studied by quasi-elastic light scattering

Physico-chemical properties of isolated zymogen granules of the mouse pancreas were studied by means of quasi-elastic light scattering. The average diameter of the granules in 0.3 M sucrose was found to be 1.1 ± 0.1 μm from the correlation time of intensity fluctuation of the scattered light. The average diameter altered depending on the osmolality of the medium in a manner that the alteration was smaller than that expected from the van't Hoff relation. Aggregation of the granules induced by the increase of Ca²⁺ concentration or the decrease of pH in the medium was also detected. The aggregation started at a critical level of 1 mM CaCl₂ or at pH 5.4.     

Elastic properties of isolated thick filaments measured by nanofabricated cantilevers.

Using newly developed nanofabricated cantilever force transducers, we have measured the mechanical properties of isolated thick filaments from the anterior byssus retractor muscle of the blue mussel Mytilus edulis and the telson levator muscle of the horseshoe crab Limulus polyphemus. The single thick filament specimen was suspended between the tip of a flexible cantilever and the tip of a stiff reference beam. Axial stress was placed on the filament, which bent the flexible cantilever. Cantilever tips were microscopically imaged onto a photodiode array to extract tip positions, which could be converted into force by using the cantilever stiffness value. Length changes up to 23% initial length (Mytilus) and 66% initial length (Limulus) were fully reversible and took place within the physiological force range. When stretch exceeded two to three times initial length (Mytilus) or five to six times initial length (Limulus), at forces approximately 18 nN and approximately 7 nN, respectively, the filaments broke. Appreciable and reversible strain within the physiological force range implies that thick-filament length changes could play a significant physiological role, at least in invertebrate muscles.     

Swimming speed distributions of bull spermatozoa as determined by quasi-elastic light scattering.

88 semen samples from 39 bulls have been investigated by the quasi-elastic light scattering technique. Normal, defective, and dead cells each yielded characteristic autocorrelation functions. The form of these functions indicates that the swimming speed distribution of normal cells is a gamma distribution with two degrees of freedom while that for defective or circular swimmers is a gamma distribution with one degree of freedom. The resulting analysis of the experimental autocorrelation functions yields the fraction of the sample that is normal, the fraction that is defective, and the average speed of each group. The average helical swimming speed of normal cells was found to be 384 micron/s, while the average trajectory speed of the circular swimmers was found to be 103 micron/s. The overall quality of the semen samples as determined by light scattering is compared to quality determination on the same samples by technicians from the artificial insemination industry.     

The internal dynamics of gene 32 protein-DNA complexes studied by quasi-elastic light scattering

The hydrodynamic properties of large homodisperse single stranded DNAs complexed with the helix destabilizing protein of phage T4, the product of gene 32 (GP32), have been measured. The results suggest a size of the binding site between 8 and 10 nucleotides/GP32 molecule, in reasonable agreement with earlier work on a complex between GP32 and single stranded 145 base DNA. From static light scattering experiments it is concluded that the persistence length of these complexes is about 30 nm, distinctly smaller than the generally accepted value for double stranded DNA. The quasi-elastic light scattering properties of the DNA-GP32 complexes were determined. The variation of the apparent translation diffusion coefficient Dapp with the scattering vector q was analyzed using the discrete ISMF and Rouse-Zimm models [S.C. Lin et al., Biopolymers 17 (1978) 425]. The model parameters that followed from the fit of Dapp versus q2 and from an extensive global analysis of the actually measured autocorrelation functions agreed with the notion that these DNA-protein complexes are indeed rather flexible. The continuous Soda model [K. Soda, Macromolecules 17 (1984) 2365] could successfully explain the variation of Dapp versus q2, assuming a persistence length of 30 nm and a base-base distance in the complex of 0.44 nm.     

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.     

Motility analysis of circularly swimming bull spermatozoa by quasi-elastic light scattering and cinematography.

The Rayleigh-Gans-Debye approximation is used to predict the electric field autocorrelation functions of light scattered from circularly swimming bull spermatozoa. Using parameters determined from cinematography and modeling the cells as coated ellipsoids of semiaxes a = 0.5 micrometers, b = 2.3 micrometers, and c = 9.0 micrometers, we were able to obtain model spectra that mimic the data exactly. A coat is found to be a necessary attribute of the particle. It is also clear that these model functions at 15 degrees may be represented by the relatively simple function used before by Hallett et al. (1978) to fit data from circularly swimming cells, thus giving some physical meaning to these functional shapes. Because of this agreement the half-widths of experimental functions can now be interpreted in terms of an oscillatory frequency for the movement of the circularly swimming cell. The cinematographic results show a trend to chaotic behavior as the temperature of the sample is increased, with concomitant decrease in overall efficiency. This is manifested by a decrease in oscillatory frequency and translational speed.     

Hydration of NaDNA by neutron quasi-elastic scattering.

Preliminary results of neutron quasi-elastic scattering experiments are reported for hydrated paracrystals of sodium deoxyribonucleic acid (NaDNA). The samples were investigated at two water contents: 3.5 +/- 1.0 and 9.5 +/- 1.5 mol H2O per mole nucleotide. The results of the scattering experiments were almost independent of whether the NaDNA fibers were oriented parallel or perpendicular to the momentum transfer. The data indicate that at the lower hydration the water molecules do not diffuse appreciably on the time scale of the neutron measurements (approximately 3 X 10(-10) s). At the higher hydration the water molecules diffuse isotropically in a sphere of 9 A in diameter with a diffusion coefficient of (5 +/- 2) X 10(-6) cm2 s-1.     

A quasi-elastic light scattering and cinematographic investigation of motile Chlamydomonas reinhardtii.

Quasi-elastic light scattering and cinematographical techniques were used to investigate the motility of Chlamydomonas reinhardtii (wild type). It was found that quantitative information on the trajectory of motion was required for a meaningful interpretation of the autocorrelation functions. Two models for describing the oscillatory motion of the cell were developed; one based on the instantaneous forward-and-backward motion of the cell, and the other based on a sinusoidal perturbation to the average forward motion. Both models gave satisfactory agreement with the shape of the experimentally measured autocorrelation function, thus making it possible to use this measurement to determine mean progressive swimming velocities in a population of greater than 200 cells.     

Rapid estimation of length distributions of microtubule preparations by quasi-elastic light scattering

A new method for determining the length distribution of microtubule preparations, using quasi-elastic light scattering (QELS) is described. The experimental electric field autocorrelation functions are analyzed using a closed-form expression recently described by Hallett, Craig, and Nickel [(1985) Biopolymers 24 , 947–960], which is incorporated into an exponential sampling procedure. The resulting length distributions are compared with those obtained for the same samples with electron microscopy (EM). If standard grid-preparation procedures were used, the EM results yielded shorter length distributions than QELS. When grids were prepared at lower microtubule number densities, less grid washing was required. In these cases, excellent agreement between the EM and QELS techniques were achieved.     

Multiple structures of thick filaments in resting cardiac muscle and their influence on cross-bridge interactions.

Based on two criteria, the tightness of packing of myosin rods within the backbone of the filament and the degree of order of the myosin heads, thick filaments isolated from a control group of rat hearts had three different structures. Two of the structures of thick filaments had ordered myosin heads and were distinguishable from each other by the difference in tightness of packing of the myosin rods. Depending on the packing, their structure has been called loose or tight. The third structure had narrow shafts and disordered myosin heads extending at different angles from the backbone. This structure has been called disordered. After phosphorylation of myosin-binding protein C (MyBP-C) with protein kinase A (PKA), almost all thick filaments exhibited the loose structure. Transitions from one structure to another in quiescent muscles were produced by changing the concentration of extracellular Ca. The probability of interaction between isolated thick and thin filaments in control, PKA-treated preparations, and preparations exposed to different Ca concentrations was estimated by electron microscopy. Interactions were more frequent with phosphorylated thick filaments having the loose structure than with either the tight or disordered structure. In view of the presence of MgATP and the absence of Ca, the interaction between the myosin heads and the thin filaments was most likely the weak attachment that precedes the force-generating steps in the cross-bridge cycle. These results suggest that phosphorylation of MyBP-C in cardiac thick filaments increases the probability of cross-bridges forming weak attachments to thin filaments in the absence of activation. This mechanism may modulate the number of cross-bridges generating force during activation.     

Enzymes in thick filaments of vertebrate cross-striated muscles]

Contemporary data on three enzymes of vertebrate cross-striated muscle thick filaments, such as creatine kinase, AMP-deaminase and phosphofructokinase, are reviewed. The physico-chemical, enzymatic and regulatory properties and localization of these enzymes in different zones of the thick filament are considered. The functional relevance of localization of creatine kinase, AMP-deaminase and phosphofructokinase on thick filaments is discussed in terms of the possible role of the enzyme adsorption on subcellular structures in regulation of metabolic processes.