A comparison of DNA compaction by arginine and lysine peptides: a physical basis for arginine rich protamines

Abstract

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.

Figure 1

Osmotic stress force curves for DNA with poly-arginine, hexa-arg⁶⁺, poly-lysine, hexa-lys⁶⁺, and poly-histidine. DNA pellets were made as described in Methods and Materials. The samples were then equilibrated against polyethylene glycol (PEG) solutions containing 10 mM Tris (pH 7.5) and 100 μM in monomer of poly-arginine , hexa-arg⁶⁺ , poly-lysine ▼, and hexa-lys⁶⁺ ■ and containing 10 mM Tris (pH 5.5) and 100 μM in monomer of poly-histidine ◆. PEG is excluded from the DNA phase and applies an osmotic pressure Π on it. The spacing between helices, D_int, was measured by x-ray scattering. The points at log(Π) ~ 5 indicate the equilibrium distance between helices in the absence of PEG. Poly-arginine, hexa-arg⁶⁺, and poly-histidine all show similar DNA-DNA force curves. Poly-lysine and hexa-lys⁶⁺ are also similar but have equilibrium spacings much larger than the others.

Figure 2

Osmotic stress force curves for DNA in ArgCl, LysCl, OrnCl, and NH₄Cl. DNA condensates prepared by ethanol precipitation and equilibrated against PEG solutions containing 10 mM Tris (pH 7.5) and two concentrations of ArgCl ●, LysCl ▲, OrnCl ■, or NH₄Cl ▼. (A): The salt concentration is 0.2M. The solid lines are fits of the data to equation (3) with λ_h = 5 Å and the Debye-Huckel shielding length at 0.2 M salt concentration λ_D = 7 Å. Electrostatics dominates at low pressures not hydration forces. (B) - The salt concentration is 1.2 M for arg+, lys⁺, orn⁺, and NH₄⁺. Force measurements for 2 M arg⁺ ( ) are also shown. The solid lines are fits of the arginine and ammonium data to a hydration force, equation (2) with λ_h = 5 Å. The overlap of the 1.2 and 2 M arg⁺ data indicates that the interactions are dominated by hydration forces at high salt concentrations, not the electrostatic repulsion seen at lower salt concentrations as in (A). The ornithine and lysine force curves show very different behavior from other univalent ions that have been examined.

Figure 2

Osmotic stress force curves for DNA in ArgCl, LysCl, OrnCl, and NH₄Cl. DNA condensates prepared by ethanol precipitation and equilibrated against PEG solutions containing 10 mM Tris (pH 7.5) and two concentrations of ArgCl ●, LysCl ▲, OrnCl ■, or NH₄Cl ▼. (A): The salt concentration is 0.2M. The solid lines are fits of the data to equation (3) with λ_h = 5 Å and the Debye-Huckel shielding length at 0.2 M salt concentration λ_D = 7 Å. Electrostatics dominates at low pressures not hydration forces. (B) - The salt concentration is 1.2 M for arg+, lys⁺, orn⁺, and NH₄⁺. Force measurements for 2 M arg⁺ ( ) are also shown. The solid lines are fits of the arginine and ammonium data to a hydration force, equation (2) with λ_h = 5 Å. The overlap of the 1.2 and 2 M arg⁺ data indicates that the interactions are dominated by hydration forces at high salt concentrations, not the electrostatic repulsion seen at lower salt concentrations as in (A). The ornithine and lysine force curves show very different behavior from other univalent ions that have been examined.

Figure 3

Osmotic stress force curves for DNA in divalent di-arginine, di-lysine, di-ornithine, and putrescine, a divalent diamine. DNA condensates were prepared by ethanol precipitation and equilibrated against PEG solutions containing 10 mM Tris (pH 7.5) and 10 mM di-arg²⁺ ●, di-lys²⁺ ▲, di-orn²⁺ ■, or putrescine²⁺ ▼. The solid lines for di-lys, di-orn, and putrescine are fits to equation (2) with the decay length λ_h fixed at 5 Å. Di-arginine shows a transition at log(Π) ~ 5.7 from a repulsive to an attractive interaction. The solid line for di-arg is the best fit to equation (4) with the decay length λ_h fixed at 5 Å and using only the data after the transition.

Figure 4

DNA-DNA interactions with +3 and higher charged lysine and ornithine peptides. (A) - Osmotic stress force curves for DNA in tri-ornithine³⁺, tetra-ornithine⁴⁺, and the tetra-amine spermine⁴⁺. DNA was precipitated by adding tri-orn³⁺, tetra-orn⁴⁺, or spermine⁴⁺ to a concentrated DNA solution in 10 mM Tris (pH 7.5) as described in Methods and Materials. The DNA pellets were then equilibrated against PEG solutions containing 10 mM Tris (pH 7.5) and 2 mM tri-orn ■, 0.5 mM tetra-orn ●, or 0.5 mM spermine ▲. The points at log(Π) ~ 5 are the equilibrium spacings, D_eq, in the absence of PEG. The solid lines are fits of the data to equation (4) with the decay length λ_h fixed at 5 Å. Note the convergence of the ornithine force curves at high pressures. (B) - Osmotic stress force curves for DNA in tri-lysine³⁺, tetra-lysine⁴⁺, and hexa-lysine⁶⁺. DNA was precipitated by adding tri-lys³⁺, tetra-lys⁴⁺, or hexa-lys⁶⁺ to a concentrated DNA solution in 10 mM Tris (pH 7.5) as described in Methods and Materials. The DNA pellets were then equilibrated against PEG solutions containing 10 mM Tris (pH 7.5) and 2 mM tri-lys³⁺ ▲, 0.5 mM tetra-lys⁴⁺ ●, or 0.1 mM hexa-lys⁶⁺ ■. The points at log(Π) ~ 5 are the equilibrium spacings, D_eq, in the absence of PEG. The solid lines are fits of the data to equation (4) with the decay length λ_h fixed at 5 Å. (C) – Normalized residuals for the osmotic stress force data shown in figures 4A and B, tetra-lys⁴⁺ ■, hexa-lys⁶⁺ ●, tri-orn³⁺ , and tetra-orn⁴⁺ . Δlog(Π(D_int)) = log(Π_exp(D_int)) − log(Π_calc(D_int)), where log(Π_exp(D_int)) is the experimental log(Π) at D_int and log(Π_calc(D_int)) is the log(Π) at D_int calculated from the double exponential fit to the data. Note the systematic deviation of the lysine peptide data from the fit.

Figure 4

DNA-DNA interactions with +3 and higher charged lysine and ornithine peptides. (A) - Osmotic stress force curves for DNA in tri-ornithine³⁺, tetra-ornithine⁴⁺, and the tetra-amine spermine⁴⁺. DNA was precipitated by adding tri-orn³⁺, tetra-orn⁴⁺, or spermine⁴⁺ to a concentrated DNA solution in 10 mM Tris (pH 7.5) as described in Methods and Materials. The DNA pellets were then equilibrated against PEG solutions containing 10 mM Tris (pH 7.5) and 2 mM tri-orn ■, 0.5 mM tetra-orn ●, or 0.5 mM spermine ▲. The points at log(Π) ~ 5 are the equilibrium spacings, D_eq, in the absence of PEG. The solid lines are fits of the data to equation (4) with the decay length λ_h fixed at 5 Å. Note the convergence of the ornithine force curves at high pressures. (B) - Osmotic stress force curves for DNA in tri-lysine³⁺, tetra-lysine⁴⁺, and hexa-lysine⁶⁺. DNA was precipitated by adding tri-lys³⁺, tetra-lys⁴⁺, or hexa-lys⁶⁺ to a concentrated DNA solution in 10 mM Tris (pH 7.5) as described in Methods and Materials. The DNA pellets were then equilibrated against PEG solutions containing 10 mM Tris (pH 7.5) and 2 mM tri-lys³⁺ ▲, 0.5 mM tetra-lys⁴⁺ ●, or 0.1 mM hexa-lys⁶⁺ ■. The points at log(Π) ~ 5 are the equilibrium spacings, D_eq, in the absence of PEG. The solid lines are fits of the data to equation (4) with the decay length λ_h fixed at 5 Å. (C) – Normalized residuals for the osmotic stress force data shown in figures 4A and B, tetra-lys⁴⁺ ■, hexa-lys⁶⁺ ●, tri-orn³⁺ , and tetra-orn⁴⁺ . Δlog(Π(D_int)) = log(Π_exp(D_int)) − log(Π_calc(D_int)), where log(Π_exp(D_int)) is the experimental log(Π) at D_int and log(Π_calc(D_int)) is the log(Π) at D_int calculated from the double exponential fit to the data. Note the systematic deviation of the lysine peptide data from the fit.

Figure 4

DNA-DNA interactions with +3 and higher charged lysine and ornithine peptides. (A) - Osmotic stress force curves for DNA in tri-ornithine³⁺, tetra-ornithine⁴⁺, and the tetra-amine spermine⁴⁺. DNA was precipitated by adding tri-orn³⁺, tetra-orn⁴⁺, or spermine⁴⁺ to a concentrated DNA solution in 10 mM Tris (pH 7.5) as described in Methods and Materials. The DNA pellets were then equilibrated against PEG solutions containing 10 mM Tris (pH 7.5) and 2 mM tri-orn ■, 0.5 mM tetra-orn ●, or 0.5 mM spermine ▲. The points at log(Π) ~ 5 are the equilibrium spacings, D_eq, in the absence of PEG. The solid lines are fits of the data to equation (4) with the decay length λ_h fixed at 5 Å. Note the convergence of the ornithine force curves at high pressures. (B) - Osmotic stress force curves for DNA in tri-lysine³⁺, tetra-lysine⁴⁺, and hexa-lysine⁶⁺. DNA was precipitated by adding tri-lys³⁺, tetra-lys⁴⁺, or hexa-lys⁶⁺ to a concentrated DNA solution in 10 mM Tris (pH 7.5) as described in Methods and Materials. The DNA pellets were then equilibrated against PEG solutions containing 10 mM Tris (pH 7.5) and 2 mM tri-lys³⁺ ▲, 0.5 mM tetra-lys⁴⁺ ●, or 0.1 mM hexa-lys⁶⁺ ■. The points at log(Π) ~ 5 are the equilibrium spacings, D_eq, in the absence of PEG. The solid lines are fits of the data to equation (4) with the decay length λ_h fixed at 5 Å. (C) – Normalized residuals for the osmotic stress force data shown in figures 4A and B, tetra-lys⁴⁺ ■, hexa-lys⁶⁺ ●, tri-orn³⁺ , and tetra-orn⁴⁺ . Δlog(Π(D_int)) = log(Π_exp(D_int)) − log(Π_calc(D_int)), where log(Π_exp(D_int)) is the experimental log(Π) at D_int and log(Π_calc(D_int)) is the log(Π) at D_int calculated from the double exponential fit to the data. Note the systematic deviation of the lysine peptide data from the fit.

Figure 5

DNA-DNA forces with mixed arginine-lysine peptides. Osmotic stress force curves for DNA in hexa-arg⁶⁺, hexa-lys⁶⁺, lys₃-arg₃⁶⁺, and (lys-arg)₃⁶⁺. DNA was precipitated by adding the +6 charged peptides to a concentrated DNA solution in 10 mM Tris (pH 7.5) as described in Methods and Materials. The DNA pellets were then equilibrated against PEG solutions containing 10 mM Tris (pH 7.5) and 100 μM hexa-lys⁶⁺ ●, hexa-arg⁶⁺ ■, lys₃-arg₃⁶⁺ ▼, or (lys-arg)₃⁶⁺ ▲. The points at log(Π) ~ 5 are the equilibrium spacings, D_eq, in the absence of PEG. The solid lines are fits of the data to equation (4) with the decay length λ_h fixed at 5 Å.

Figure 6

Dependence of the interaction free energies on peptide length N. (A) – The free energy of interaction (in units of kT/bp) at the equilibrium spacing, D_eq, is calculated from the double exponential fits to the osmotic stress data and equation (7) for the arginine , ornithine , and lysine peptides. The much weaker attraction between DNA helices with lysine and ornithine peptides compared with arginine is apparent. (B) – The attractive and repulsive free energy components at 25 Å (in units of kT/bp) of the interaction are calculated from the double exponential fits to the osmotic stress force curves and equations (5) and (6). The repulsive, ΔG_R(25 Å)/kT, and attractive, ΔG_A(25 Å)/kT, free energies, respectively, are shown for the arginine ( , ◆), ornithine ( , ■) and lysine ( , ●) peptides.

Figure 6

Dependence of the interaction free energies on peptide length N. (A) – The free energy of interaction (in units of kT/bp) at the equilibrium spacing, D_eq, is calculated from the double exponential fits to the osmotic stress data and equation (7) for the arginine , ornithine , and lysine peptides. The much weaker attraction between DNA helices with lysine and ornithine peptides compared with arginine is apparent. (B) – The attractive and repulsive free energy components at 25 Å (in units of kT/bp) of the interaction are calculated from the double exponential fits to the osmotic stress force curves and equations (5) and (6). The repulsive, ΔG_R(25 Å)/kT, and attractive, ΔG_A(25 Å)/kT, free energies, respectively, are shown for the arginine ( , ◆), ornithine ( , ■) and lysine ( , ●) peptides.