Search

Short arginine-rich proteins called protamines mediate the near crystalline DNA packaging in most vertebrate sperm cells. Protamines are synthesized during spermiogenesis and condense the paternal genome into a transcriptionally inactive state in late-stage spermatids. Protamines from eutherian mammals, including bulls and humans, also contain multiple cysteine residues that form intra- and interprotamine sulfur-sulfur bonds during the final stages of sperm maturation. Although the cross-linked protamine network is known to stabilize the resulting nucleoprotamine structure, little is known about the role of disulfide bonds on DNA condensation in the mammalian sperm. Using small angle x-ray scattering, we show that isolated bull nuclei achieve slightly lower DNA packing densities compared to salmon nuclei despite salmon protamine lacking cysteine residues. Surprisingly, reduction of the intermolecular sulfur-sulfur bonds of bull protamine results in tighter DNA packing. Complete reduction of the intraprotamine disulfide bonds ultimately leads to decondensation, suggesting that disulfide-mediated secondary structure is also critical for proper protamine function. Lastly, comparison of multiple bull collections showed some to have aberrant x-ray scattering profiles consistent with incorrect disulfide bond formation. Together, these observations shed light on the biological functions of disulfide linkages for in vivo DNA packaging in sperm chromatin.

Structured water on apposing surfaces can generate significant energies due to reorganization and displacement of water as the surfaces encounter each other. Force measurements on a multitude of biological structures using the osmotic stress technique have elucidated commonalities that point toward an underlying hydration force. In this review, the forces of two contrasting systems are considered in detail: highly charged DNA and nonpolar, uncharged hydroxypropyl cellulose. Conditions for both net repulsion and attraction, along with the measured exclusion of chemically different solutes from these macromolecular surfaces, are explored and demonstrate common features consistent with a hydration force origin. Specifically, the observed interaction forces can be reduced to the effects of perturbing structured surface water.

Understanding the strength and specificity of interactions among biologically important macromolecules that control cellular functions requires quantitative knowledge of intermolecular forces. Controlled DNA condensation and assembly are particularly critical for biology, with separate repulsive and attractive intermolecular forces determining the extent of DNA compaction. How these forces depend on the charge of the condensing ion has not been determined, but such knowledge is fundamental for understanding the basis of DNA-DNA interactions. Here, we measure DNA force-distance curves for a homologous set of arginine peptides. All forces are well fit as the sum of two exponentials with 2.4- and 4.8-Å decay lengths. The shorter-decay-length force is always repulsive, with an amplitude that varies slightly with length or charge. The longer-decay-length force varies strongly with cation charge, changing from repulsion with Arg1 to attraction with Arg2. Force curves for a series of homologous polyamines and the heterogeneous protein protamine are quite similar, demonstrating the universality of these forces for DNA assembly. Repulsive amplitudes of the shorter-decay-length force are species-dependent but nearly independent of charge within each species. A striking observation was that the attractive force amplitudes for all samples collapse to a single curve, varying linearly with the inverse of the cation charge.

Many specific sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically, then by linearly diffusing or hopping along DNA until either the protein dissociates from the DNA or it finds the recognition sequence. We have devised a method for measuring one-dimensional diffusion along DNA based on the ratio of the dissociation rate of protein from DNA fragments containing one specific binding site to the dissociation rate from DNA fragments containing two specific binding sites. Our extensive measurements of dissociation rates and specific-nonspecific relative binding constants of the restriction nuclease EcoRI enable us to determine the diffusion rate of nonspecifically bound protein along the DNA. By varying the distance between the two binding sites, we confirm a linear diffusion mechanism. The sliding rate is relatively insensitive to salt concentration and osmotic pressure, indicating that the protein moves smoothly along the DNA probably following the helical phosphate-sugar backbone of DNA. We calculate a diffusion coefficient for EcoRI of 3 x 10(4) bp(2) s(-)(1) EcoRI is able to diffuse approximately 150 bp, on average, along the DNA in 1 s. This diffusion rate is about 2000-fold slower than the diffusion of free protein in solution. A factor of 40-50 can be accounted for by rotational friction resulting from following the helical path of the DNA backbone. Two possibilities could account for the remaining activation energy: salt bridges between the DNA and the protein are transiently broken, or the water structure at the protein-DNA interface is disrupted as the two surfaces move past each other.

By combining single-molecule magnetic tweezers and osmotic stress on DNA assemblies, we separate attractive and repulsive components of the total intermolecular interaction between multivalent cation condensed DNA. Based on measurements of several different cations, we identify two invariant properties of multivalent cation-mediated DNA interactions: repulsive forces decay exponentially with a 2.3 +/- 0.1 A characteristic decay length and the attractive component of the free energy is always 2.3 +/- 0.2 times larger than the repulsive component of the free energy at force-balance equilibrium. These empirical constraints are not consistent with current theories that attribute DNA-DNA attractions to a correlated lattice of counterions. The empirical constraints are consistent with theories for Debye-Hückel interactions between helical line charges and with the order-parameter formalism for hydration forces. Each of these theories posits exponentially decaying attractions and, if we assume this form, our measurements indicate a cation-independent, 4.8 +/- 0.5 A characteristic decay length for intermolecular attractions between condensed DNA molecules.

Interactions governing protein folding, stability, recognition, and activity are mediated by hydration. Here, we use small-angle neutron scattering coupled with osmotic stress to investigate the hydration of two proteins, lysozyme and guanylate kinase (GK), in the presence of solutes. By taking advantage of the neutron contrast variation that occurs upon addition of these solutes, the number of protein-associated (solute-excluded) water molecules can be estimated from changes in both the zero-angle scattering intensity and the radius of gyration. Poly(ethylene glycol) exclusion varies with molecular weight. This sensitivity can be exploited to probe structural features such as the large internal GK cavity. For GK, small-angle neutron scattering is complemented by isothermal titration calorimetry with osmotic stress to also measure hydration changes accompanying ligand binding. These results provide a framework for studying other biomolecular systems and assemblies using neutron scattering together with osmotic stress.

Using the osmotic stress technique together with a self-cleavage assay we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers of water molecules released coupled to specific complex formation. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes used to set water activity. The observed osmotic dependence indicates that the nonspecific DNA-BamHI complex sequesters some 120-150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity suggests that these waters are sterically inaccessible to solutes. This result is in close agreement with differences in the structures determined by x-ray crystallography. We demonstrate additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent (with up to 10-fold difference between solutes). This result is expected for reactions resulting in a large change in a surface exposed area.

We use the osmotic pressure dependence of dissociation rates and relative binding constants to infer differences in sequestered water among complexes of lambda Cro repressor with varied DNA recognition sequences. For over a 1000-fold change in association constant, the number of water molecules sequestered by non-cognate complexes varies linearly with binding free energy. One extra bound water molecule is coupled with the loss of approximately 150 cal/mol complex in binding free energy. Equivalently, every tenfold decrease in binding constant at constant salt and temperature is associated with eight to nine additional water molecules sequestered in the non-cognate complex. The relative insensitivity of the difference in water molecules to the nature of the osmolyte used to probe the reaction suggests that the water is sterically sequestered. If the previously measured changes in heat capacity for lambda Cro binding to different non-cognate sequences are attributed solely to this change in water, then the heat capacity change per incorporated water is almost the same as the difference between ice and water. The associated changes in enthalpies and entropies, however, indicate that the change in complex structure involves more than a simple incorporation of fixed water molecules that act as adaptors between non-complementary surfaces.

The physical forces that underlie the exclusion of solutes from macromolecular surfaces can be probed in a similar way as the measurement of forces between macromolecules in condensed arrays using the osmotic stress technique and x-ray scattering. We report here the dependence of alcohol exclusion or, equivalently, the preferential hydration of DNA on the spacing between helices in condensed arrays. The actual forces describing exclusion are quite different from the commonly assumed steric crowding coupled with weak binding. For a set of 12 nonpolar alcohols, exclusion is due to repulsive hydration interactions with the charged DNA surface. Exclusion amplitudes do not depend simply on size, but rather on the balance between alkyl carbons and hydroxyl oxygens. Polyols are included at very close spacings. The distance dependence of polyol inclusion, however, is quite different from nonpolar alcohol exclusion, suggesting the underlying mechanism of interaction is different.

We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in order to distinguish the contributions of these solution components to specific and non-specific binding. For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along non-specific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and the dissociation of non-specifically bound protein. We demonstrated previously that the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and non-specific binding that is independent of the osmolyte nature. The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area. In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies. We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay. The observed weak salt-sensitivity of the ratio of specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action. The seeming lack of a dependence on viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.

The forces that govern DNA double helix organization are being finally systematically measured. The non-specific longer-range interactions--such as electrostatic interactions, hydration, and fluctuation forces--that treat DNA as a featureless rod are reasonably well recognized. Recently, specific interactions--such as those controlled by condensing agents or those consequent to helical structure-are beginning to be recognized, quantified and tested.

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin II minifilaments is dependent both on Mg2+ concentration and on the state of phosphorylation of three serine sites at the C-terminal end of the heavy chains. Previous electric birefringence experiments on minifilaments showed a large dependence of signal amplitude on the phosphorylation state and Mg2+ concentration, consistent with large changes in filament flexibility. These observations suggested that minifilament stiffness was important for function. We now report that the binding of nucleotides to dephosphorylated minifilaments at Mg2+ concentrations needed for optimal activity increases the flexibility by about 10-fold, as inferred from the birefringence signal amplitude increase. An increase in flexibility with nucleotide binding is not observed for dephosphorylated minifilaments at lower Mg2+ concentrations or for phosphorylated minifilaments at any Mg2+ concentrations examined. The relaxation times for minifilament rotations that are sensitive to the conformation myosin heads are also observed to depend on phosphorylation, Mg2+ concentration, and nucleotide binding. These latter experiments indicate that the actin-activated Mg2+ concentration, and nucleotide binding. These latter experiments indicate that the actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin II correlates with both changes in myosin head conformation and the ability of minifilaments to cycle between stiff and flexible conformations coupled to nucleotide binding and release.

Simultaneous measurements on the packing and energetics of high-density liquid crystalline DNA phases show that the crowding of long DNA polyelectrolytes at ever increasing concentrations is accomplished through straightening of the random coils that the double helix assumes in dilute solution. X-ray scattering by ordered phases reveals that the local straightening of the molecules is also accompanied by their progressive immobilization and confinement within the molecular 'cages' created by neighboring molecules. These effects can be clearly observed through the measured energies of DNA packing under osmotic stress and through the changes in structural and dynamic characteristics of X-ray scattering from DNA in ordered arrays at different concentrations. The character of the confinement of large DNA motions for a wide range of DNA concentrations is dominated by the soft potentials of direct interaction. We do not see the power-law variation of energy vs. volume expected from space-filling fluctuations of molecules that enjoy no interaction except the hard clash of steric repulsion. Rather, in highly concentrated DNA mesophases we see a crowding of molecules through electrostatic or hydration repulsion that confines their movements and positions. This view is based on directly measured packing energies as well as on concurrently measured structural parameters while the DNA double helices are condensed under an externally applied osmotic pressure.

The electrophoretic mobility shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA-protein interactions. The technique uses polyacrylamide gel electrophoresis to separate DNA-protein or RNA-protein complexes from free DNA or RNA. Polyacrylamide gels stabilize DNA-protein, RNA-protein, or protein-protein complexes by a crowding or caging mechanism. Still every technique has its limitations. EMSA, as standardly practiced today, works well for complexes with association binding constants Ka>109 M−1 under normal conditions of salt and pH. Many DNA-protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. We take advantage of our previous observation that neutral osmolytes can strongly slow down the rate of DNA-protein complex dissociation to develop a method that uses osmotic stress to stabilize complexes in the gel matrix as well as in the solution. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using the electrophoretic mobility shift assay. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein-DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and capillary electrophoresis is straightforward.

In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. We have previously observed that the net attraction between salmon protamine condensed DNA helices was much smaller for DNA condensed by the equivalent homo-arginine peptide. We hypothesized that this is caused by the neutral amino acids present in protamines. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. The component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance have been determined by the osmotic stress technique coupled with x-ray scattering as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short-range repulsion; while only slightly decreasing the longer-ranged attraction. The decrease in net attraction between salmon protamine condensed helices compared with arginine homopeptides can be well explained by amino acid content alone. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both these observation have biological implications for protamine-DNA packaging in sperm heads.

Directly measured forces between DNA helices in ordered arrays have been reduced to simple force coefficients and mathematical expressions for the interactions between pairs of molecules. The tabulated force parameters and mathematical expressions can be applied to parallel molecules or, by transformation, to skewed molecules of variable separation and mutual angle. This "toolbox" of intermolecular forces is intended for use in modelling molecular interactions, assembly, and conformation. The coefficients characterizing both the exponential hydration and the electrostatic interactions depend strongly on the univalent counterion species in solution, but are only weakly sensitive to anion type and temperature (from 5 to 50 degrees C). Interaction coefficients for the exponentially varying hydration force seen at spacings less than 10 to 15 A between surfaces are extracted directly from pressure versus interaxial distance curves. Electrostatic interactions are only observed at larger spacings and are always coupled with configurational fluctuation forces that result in observed exponential decay lengths that are twice the expected Debye-Huckel length. The extraction of electrostatic force parameters relies on a theoretical expression describing steric forces of molecules "colliding" through soft exponentially varying direct interactions.

We have applied a self-cleavage assay, developed previously by us, to measure EcoRV-DNA binding in solution. Self-cleavage assay monitors only enzymatically competent complexes of the endonuclease. This technique does not have the limitations of more commonly used gel mobility shift assay while providing the same level of sensitivity. We found that the EcoRV has quite unusual kinetics of specific complex formation in the absence of divalent ions that was not reported previously. A significant fraction of the total enzyme, ∼ 45%, forms enzymatically competent complexes unusually slowly, especially at pH 7.6. This novel result can be explained by a very slow transition between two conformations of the free enzyme in solution. The equilibrium distribution of the slowly and quickly associating protein structures and their exchange kinetics may depend on many parameters including pH, salt, osmolytes, and divalent cations. The observation of at least two kinetics components in association indicates that EcoRV is an allosteric protein with at least two conformations. Allosterism is now recognized as important concept for DNA-protein complexes, offering an additional level of control over binding and activity. We are continuing our investigation into the EcoRV structures responsible for the different kinetic classes of association.

The actin-activated Mg(2+)-ATPase activity of filamentous Acanthamoeba myosin II is regulated by the state of phosphorylation of three sites at the C terminus of each heavy chain. This phosphorylation at the tip of the tails of monomers in a bipolar filament abolishes the activity of sites some 90 nm distant in the globular heads. Previous studies with copolymeric filaments of phosphorylated and dephosphorylated monomers strongly indicated that the activity of each monomer in a filament is dependent on the level of phosphorylation of neighboring monomers in the filament. We report here electric birefringence measurements showing that, although the overall structures of phosphorylated and dephosphorylated filaments are very similar, large, Mg2+ concentration-dependent differences in internal motion and flexibility are observed. Filaments of dephosphorylated myosin II appear to be about 50-fold stiffer than filaments of phosphorylated myosin II at 4 mM Mg2+. These results are consistent with a model in which the stiffness of the putative hinge region within the rod-like tail of each monomer is determined by the phosphorylation state of the C-terminal tails of overlapping, neighboring monomers. The flexibility of the filaments appears to be directly related to their actin-activated Mg(2+)-ATPase activity.

Binding of Zn 2+ to the 5 S RNA gene sequence of Xenopus borealis results in strong bending of the DNA, as inferred from transient electric birefringence data. The effect is specific for Zn 2+ ; several other divalent ions are not able to induce a bend of a similar magnitude. Using five different fragments that span the binding sequence, we are able to estimate a bend magnitude of at least 55 ° centered at base-pair +65 within the gene. This places the bend within the binding domain of the gene-regulatory protein transcription factor (TF) IIIA. Recent evidence has shown that the protein-DNA complex is also bent. Although our data do not allow us directly to link the two bends, our results suggest that TFIIIA could form a folded structure by stabilizing the same bent conformation that is induced by binding of Zn 2+ . The chemistry of Zn 2+ binding to DNA, and the sequence around the bend center, suggest that the bend is most probably caused by joint co-ordination of Zn 2+ to the N-7 groups of stacked purine residues.

DNA in vertebrate sperm nuclei is organized differently from any other cell. DNA is compacted in tightly packed (∼ 400 mg/ml) hexagonal arrays. Since DNA repair is absent in sperm, dense packaging of DNA is considered necessary to protect against damage by reactive oxidizing species. DNA damage correlates with infertility and likely contributes to miscarriages and birth defects. Our goal is to characterize protamine defects that result in DNA mispackaging. Small (∼30-50 amino acids), arginine rich (50-70%) peptides called protamines assemble DNA in sperm nuclei. Protamines will spontaneously condense DNA from dilute solution into close-packed, liquid crystalline arrays. Both sperm nuclei and reconstituted protamine-DNA arrays are sufficiently ordered that the interhelical spacing or packing density can be determined by x-ray scattering.Insufficient protamine to neutralize all DNA has been linked to excess damage. Salmon sperm nuclei can be fractionated on sucrose density gradients. The interhelical scattering maxima of the heavy and light fractions overlap indicating that all nuclei have a tightly packed fraction of DNA. The lighter fraction, however, shows a scattering profile that is skewed toward larger spacings. Part of the DNA is not as well packed as the majority. Adding excess protamine to the light fraction recovers the scattering profile of the heavy fraction.Protamines are initially serine phosphorylated when replacing histones on DNA. Incomplete dephosphorylation has also been linked to DNA damage. Herring protamines can be partially phosphorylated with protein kinase A. We find that even modest phosphorylation that reduces the protamine charge by only 5% will increase the water volume accessible to oxidizing species in reconstituted DNA-protamine assemblies by 50%.

Pressurized DNA storage and pressure-driven DNA ejection in bacteriophage lambda provide an attractive model system for investigating DNA packaging in tight spaces. Moreover, lambda phage holds therapeutic promise as an alternative to antibiotics and vehicle for gene delivery. A wide variety of experimental and theoretical tools have been employed to interrogate DNA mechanics in lambda phage. Normally triggered ejection leads to loss of all DNA from the phage head; partial DNA ejection can be achieved by either counteracting the DNA pressure with external osmotic pressure or reducing the DNA-DNA repulsion with multivalent cations. Our objective in this study was to systematically map the lengths of DNA left inside partially ejected lambda phage. We determined the expected length of DNA inside the phage by monitoring the amount of ejected DNA with UV spectroscopy and assayed the actual length left directly with field-inversion gel electrophoresis. Previously used experimental methods, such as UV spectroscopy, were examined and used to assess the results from our modified protocols. Surprisingly, we found that lengths of retained DNA were concentrated around multiple specific lengths rather than the single equilibrium length that would fit thermodynamic models of DNA packaging in lambda phage. These DNA lengths occurred at regular intervals and highlighted unexpected conformational variants, sequence specific interactions, or discontinuities in DNA packaging. Structurally relevant patterns were observed when cobalt hexamine, magnesium chloride, spermine, and bis(ethyl) spermine were used as counterions. Taken together, our results show that thermodynamic models of DNA packaging in lambda phage need to be reevaluated to account for the size specific ejection that has been observed in our study.

Biological systems under extreme conditions often resort to unusual structures to achieve special functions for survival. The “bioengineering” principles of such “extreme” structures may inspire biomimic designs of functional materials. Here we give several examples of such naturally occurring unusual structures probed with x-ray scattering. i) In bacterial spores, the spore coat appears to comprise of laminar layers of quasi-2D crystals with periodicity of ∼1 nm. Such ordered assembly may be responsible for the spore resistance to heat, toxic chemicals, and mechanical disruption. ii) In starved bacterial cells, the DNA packaging protein (DPS) is over-produced to compact the chromosomal DNA close to crystalline density to protect the genomic integrity and facilitate homologous recombination. iii) In bacterial cells with over-produced DNA, mild treatment with antibiotics can lead to liquid-crystalline DNA that responds to external osmotic stress. Future work includes 1) uncovering the molecular basis of characterized structures and 2) extending into systems such as cells under radiation or heat and cancer cells.

We have analyzed the structure of complexes between DNA gyrase and four defined DNA fragments by electric dichroism. Both the extrapolated dichroism and relaxation time of these complexes suggest that a single turn of DNA is wrapped around the enzyme with the entry and exit points located close together. The average angle between the DNA tails emerging from the particle is about 120 degrees. This structure is consistent with that seen by electron microscopy. Addition of ATP or the non-hydrolyzable ATP analog 5'-adenylyl-beta, gamma-imidodiphosphate results in a structural change of the complex, consistent with the DNA tails now being wrapped around the protein. The significance of these observations with respect to the mechanism of DNA supercoiling by DNA gyrase is discussed.

In order to test the diffuse ion atmosphere polarization model recently developed by us. the effects of ionic strength, titrating with Mg2+ and CO(NH)63+, and coion charge on the electric polarizability of short fragments of DNA are investigated. The results are consistent with the predictions of the theory and show that the diffuse ion atmosphere polarization contributes significantly to the overall orientation of DNA. At low ionic strengths, we attempt to separate the total dipole moment into two components: one that agrees well with the Debye-Huckel ion atmosphere calculations, while the other, presumably due to condensed counlerion polarization, appears to be substantially independent of the ionic strength. At higher salt concentrations. however, a simple separation into dipole components is not possible, perhaps due to a significant coupling of ion flows between the diffuse atmosphere and the condensed counterion layer.

After removal of the 66 COOH-terminal amino acids from each of its two heavy chains by chymotrypsin digestion, Acanthamoeba myosin II forms only parallel dimers under conditions in which native myosin II forms bipolar filaments (Kuznicki, J., Cote, G. P., Bowers, B., and Korn, E. D. (1985) J. Biol. Chem. 260, 1967-1972). We have studied the solution structure of the chymotrypsin-cleaved myosin II by electric birefringence. Only two species, known to be monomer and parallel dimer from previous studies, were detected. The contribution to the birefringence decay from dimer increased from about 10 to 70% as the KCl concentration was lowered from 100 mM to 0 in 50% glycerol. At all ionic strengths, the monomer had a relaxation time corrected to water at 20 degrees C of 8.2 microseconds, whereas a relaxation time of 10.3 microseconds was expected for monomers with straight rigid rods. This strongly indicates that the myosin rod in solution is bent. On the assumption that there is a single bend 26 nm from the tip of the tail, as suggested by electron microscopy, it was calculated that the average bend angle would be 110 degrees, in solution, if as seems most likely, the average angle between the two globular heads were 180 degrees. The observed relaxation time of the dimer corrected to water at 20 degrees C was 25 microseconds, independent of ionic strength, which, if the motion of the heads were unrestricted, is consistent with a structure for a parallel dimer in which either the two monomer subunits have straight rigid rods and are staggered by about 28 nm or only one is bent and the stagger is 30 nm. As described in the accompanying Appendix, either of these dimers can be assembled into a bipolar filament compatible with the apparent structure of filaments of native myosin II (Pollard, T.D. (1982) J. Cell Biol. 95, 816-825).

We have used electric dichroism to study the arrangement of nucleosomes in 30 nm chromatin solenoidal fibers prepared from a variety of sources (CHO cells, HeLa cells, rat liver, chicken erythrocytes, and sea urchin sperm) in which the nucleosome spacer length varies from approximately 10 to approximately 80 bp. Field-free relaxation times are consistent only with structures containing 6 +/- 1 nucleosomes for every 11 nm of solenoidal length. With very few assumptions about the arrangement of the spacer DNA, our dichroism data are consistent with the same orientation of the chromatosomes for every chromatin sample examined. This orientation, which maintains the faces of the radially arranged chromatosomes inclined at an angle between 20 degrees-33 degrees to the solenoid axis, thus appears to be a general structural feature of the higher order chromatin fiber.