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.

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%.

Nat. Chem. Biol. 10, 861–867 (2014); published online 7 September 2014; corrected after print 4 November 2014 In the version of this article initially published, a callout made to Figure 3a should have referred to Figure 4a in the first paragraph of the subsection entitled “Solid-to-fluid DNA transition inside the HSV-1 capsid” on page 864.

Nat. Chem. Biol. 10, 861–867 (2014); published online 7 September 2014; corrected after print 4 November 2014 In the version of this article initially published, a callout made to Figure 3a should have referred to Figure 4a in the first paragraph of the subsection entitled “Solid-to-fluid DNA transition inside the HSV-1 capsid” on page 864.

It is widely accepted that the interaction between lipid bilayers at closest separation is dominated by a repulsive hydration force arising from the structuring of water molecules on the lipid bilayer surface. This force has been recognized as a major activation energy barrier preventing fusion of bilayers. Our working hypothesis is that the orientation of the hydrogen bond network on the bilayer surface determines the amplitude of the hydration force. We are testing this hypothesis using the 1,2-dioleoyl-sn-glycero-3-phospho-[1′-myo-inositol-4',5′-bisphosphate](PI(4,5)P2) lipid and its isomers which differ in the position of phosphate groups on the inositol ring. Phosphatidylinositol-bisphosphate (PIP2) lipids are pivotal in signaling and play an important role in exocytosis. Specifically, we have utilized small angle X-ray diffraction (SAXS) and monitored the structural consequences of osmotic pressure in multilamellar suspensions of dioleoylphosphatidylcholine (DOPC) in the presence of PI(4,5)P2. Our preliminary data (powder X-ray diffraction and reconstructed electron density profiles) show that there are notable changes in bilayer structure, particularly the lamellar repeat distance, thickness, and forces in the presence of PI(4,5)P2. We are currently investigating whether the effects of PIP2 lipids are dependent on the phosphate group position, ionic strength, and temperature. Since the relative position of the hydrogen bond acceptor in phosphates are determined by their exact molecular structure, our results will be relevant in the context of molecular recognition involving PIP2 lipids.

Significance The efficiency of viral replication is limited by the ability of the virus to eject its genome into a cell. We discovered a fundamentally important mechanism for translocation of viral genomes into cells. For the first time, to our knowledge, we show that tightly packaged DNA in the viral capsid of a bacterial virus (phage λ) undergoes a solid-to-fluid–like structural transition that facilitates infection close to 37 °C. Our finding shows a remarkable physical adaptation of bacterial viruses to the environment of Escherichia coli cells in a human host.

Type II restriction endonucleases require metal ions to specifically cleave DNA at canonical sites. Despite the wealth of structural and biochemical information, the number of Mg(2+) ions used for cleavage by EcoRV, in particular, at physiological divalent ion concentrations has not been established. In this work, we employ a single-turnover technique that uses osmotic stress to probe reaction kinetics between an initial specific EcoRV-DNA complex formed in the absence of Mg(2+) and the final cleavage step. With osmotic stress, complex dissociation before cleavage is minimized and the reaction rates are slowed to a convenient time scale of minutes to hours. We find that cleavage occurs by a two-step mechanism that can be characterized by two rate constants. The dependence of these rate constants on Mg(2+) concentration and osmotic pressure gives the number of Mg(2+) ions and water molecules coupled to each kinetic step of the EcoRV cleavage reaction. Each kinetic step is coupled to the binding 1.5-2.5 Mg(2+) ions, the uptake of ∼30 water molecules, and the cleavage of a DNA single strand. We suggest that each kinetic step reflects an independent, rate-limiting conformational change of each monomer of the dimeric enzyme that allows Mg(2+) ion binding. This modified single-turnover protocol has general applicability for metalloenzymes.

Protamines are small, highly positively charged peptides used to package DNA at very high densities in sperm nuclei. Tight DNA packing is considered essential for the minimization of DNA damage by mutagens and reactive oxidizing species. A striking and general feature of protamines is the almost exclusive use of arginine over lysine for the positive charge to neutralize DNA. We have investigated whether this preference for arginine might arise from a difference in DNA condensation by arginine and lysine peptides. The forces underlying DNA compaction by arginine, lysine, and ornithine peptides are measured using the osmotic stress technique coupled with X-ray scattering. The equilibrium spacings between DNA helices condensed by lysine and ornithine peptides are significantly larger than the interhelical distances with comparable arginine peptides. The DNA surface-to-surface separation, for example, is some 50% larger with polylysine than with polyarginine. DNA packing by lysine rich peptides in sperm nuclei would allow much greater accessibility to small molecules that could damage DNA. The larger spacing with lysine peptides is caused by both a weaker attraction and a stronger short-range repulsion relative to that of the arginine peptides. A previously proposed model for binding of polyarginine and protamine to DNA provides a convenient framework for understanding the differences between the ability of lysine and arginine peptides to assemble DNA.

The DNA structure in phage capsids is determined by DNA-DNA interactions and bending energy. The effects of repulsive interactions on DNA interaxial distance were previously investigated, but not the effect of DNA bending on its structure in viral capsids. By varying packaged DNA length and through addition of spermine ions, we transform the interaction energy from net repulsive to net attractive. This allowed us to isolate the effect of bending on the resulting DNA structure. We used single particle cryo-electron microscopy reconstruction analysis to determine the interstrand spacing of double-stranded DNA encapsidated in phage lambda capsids. The data reveal that stress and packing defects, both resulting from DNA bending in the capsid, are able to induce a long-range phase transition in the encapsidated DNA genome from a hexagonal to a cholesteric packing structure. This structural observation suggests significant changes in genome fluidity as a result of a phase transition affecting the rates of viral DNA ejection and packaging.

Direct transfer of proteins between DNA helices is a recognized important feature of the recognition site search process. Direct transfer is characterized by a dissociation rate that depends on total DNA concentration. This is taken as evidence for the formation of an intermediate DNA-protein-DNA ternary complex. We find that the dissociation rate of EcoRI-DNA-specific complexes at 80 mM NaCl depends on the concentration of competitor oligonucleotide suggesting that direct transfer contributes to EcoRI dissociation. This dependence on competitor DNA concentration is not seen at 180 mM salt. A careful examination of the salt concentration dependence of the dissociation rate, however, shows that the predictions for the formation of a ternary complex are not observed experimentally. The findings can be rationalized by considering that just after dissociating from a DNA fragment the protein remains in close proximity to that fragment, can reassociate with it, and diffuse back to the recognition site rather than bind to an oligonucleotide in solution, a hopping excursion. The probability that a protein will bind to an oligonucleotide during a hop can be approximately calculated and shown to explain the data. A dependence of the dissociation rate of a DNA-protein complex on competitor DNA concentration does not necessarily mean direct transfer.

Trimethylamine N-oxide (TMAO) is a naturally occurring protecting osmolyte that stabilizes the folded state of proteins and also counteracts the destabilizing effect of urea on protein stability. Experimentally, it has been inferred that TMAO is preferentially excluded from the vicinity of protein surfaces. Here, we combine computer modeling and experimental measurements to gain an understanding of the mechanism of the protecting effect of TMAO on proteins. We have developed an all-atom molecular model for TMAO that captures the exclusion of TMAO from model compounds and protein surfaces, as a consequence of incorporating realistic TMAO-water interactions through osmotic pressure measurements. Osmotic pressure measurements also suggest no significant attraction between urea and TMAO molecules in solution. To obtain an accurate potential for molecular simulations of protein stability in TMAO solutions, we have explored different ways of parametrizing the protein/osmolyte and osmolyte/osmolyte interactions by scaling charges and the strength of Lennard-Jones interactions and carried out equilibrium folding experiments of Trp-cage miniprotein in the presence of TMAO to guide the parametrization. Our calculations suggest a general principle for preferential interaction behavior of cosolvents with protein surfaces--preferentially excluded osmolytes have repulsive self-interaction given by osmotic coefficient φ > 1, while denaturants, in addition to having attractive interactions with the proteins, have favorable self-interaction given by osmotic coefficient φ < 1, to enable preferential accumulation in the vicinity of proteins.

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.

The DNA binding stringency of restriction endonucleases is crucial for their proper function. The X-ray structures of the specific and non-cognate complexes of the restriction nuclease EcoRV are considerably different suggesting significant differences in the hydration and binding free energies. Nonetheless, the majority of studies performed at pH 7.5, optimal for enzymatic activity, have found a < 10-fold difference between EcoRV binding constants to the specific and nonspecific sequences in the absence of divalent ions. We used a recently developed self-cleavage assay to measure EcoRV-DNA competitive binding and to evaluate the influence of water activity, pH and salt concentration on the binding stringency of the enzyme in the absence of divalent ions. We find the enzyme can readily distinguish specific and nonspecific sequences. The relative specific-nonspecific binding constant increases strongly with increasing neutral solute concentration and with decreasing pH. The difference in number of associated waters between specific and nonspecific DNA-EcoRV complexes is consistent with the differences in the crystal structures. Despite the large pH dependence of the sequence specificity, the osmotic pressure dependence indicates little change in structure with pH. The large osmotic pressure dependence means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowded environment of the cell. In addition to divalent ions, water activity and pH are key parameters that strongly modulate binding specificity of EcoRV.

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. 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. We have previously observed that the neutral amino acids in salmon protamine decrease the net attraction between protamine-DNA helices compared with the equivalent homo-arginine peptide. Using osmotic stress coupled with x-ray scattering, we have investigated the component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance 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 affecting longer range attraction. The amino acid content alone of salmon protamine is enough to rationalize the forces that package DNA in sperm heads. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both of these observations have biological implications for protamine-DNA packaging in sperm heads.

Using osmotic stress coupled with X-ray scattering, we have directly examined the salt sensitivity of the intermolecular forces between helices in condensed protamine-DNA arrays. Thermodynamic forces are measured from the dependence of DNA helical interaxial spacings on external salt concentration or the osmotic pressure applied by neutral polymer solutions in equilibrium with the condensed phase. Force curves of salmon protamine-DNA condensates are highly dependent on salt species and concentration, indicating salt binding to protamine-DNA complexes. This dependence of the forces on salt species follows the Hofmeister series for anions. Chaotropic anions bind more tightly to protamine-DNA arrays than kosmotropic anions, thus more greatly disrupting the attractive thermodynamic forces. Variations with cation type are small compared with those observed for anions. Further, osmotic stress is used to estimate the number of ions bound in the condensed phase through a Gibbs-Duhem relationship. We estimate that at equilibrium, ∼1 Br(-) is bound per protamine molecule at 200 mM NaBr concentration. Remarkably, this one bound anion results in a change of ∼12% in the surface-to-surface distance between DNA helices. Potential biological implications of this attractive force salt sensitivity are discussed.

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.

Using solution synchrotron x-ray scattering, we measure the variation of DNA-DNA d spacings in bacteriophage λ with mono-, di-, and polyvalent salt concentrations, for wild-type [48.5×10(3) base pairs (bp)] and short-genome-mutant (37.8 kbp) strains. From the decrease in d spacings with increasing salt, we deduce the relative contributions of DNA self-repulsion and bending to the energetics of packaged phage genomes. We quantify the DNA-DNA interaction energies within the intact phage by combining the measured d spacings in the capsid with measurements of osmotic pressure in DNA assemblies under the same salt conditions in bulk solution. In the commonly used Tris-Mg buffer, the DNA-DNA interaction energies inside the phage capsids are shown to be about 1kT/bp, an order of magnitude larger than the bending energies.

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.