Determining the Locations of Ions and Water around DNA from X-Ray Scattering Measurements

Abstract

Nucleic acids carry a negative charge, attracting salt ions and water. Interactions with these components of the solvent drive DNA to condense, RNA to fold, and proteins to bind. To understand these biological processes, knowledge of solvent structure around the nucleic acids is critical. Yet, because they are often disordered, ions and water evade detection by x-ray crystallography and other high-resolution methods. Small-angle x-ray scattering (SAXS) is uniquely sensitive to the spatial correlations between solutes and the surrounding solvent. Thus, SAXS provides an experimental constraint to guide or test emerging solvation theories. However, the interpretation of SAXS profiles is nontrivial because of the difficulty in separating the scattering signals of each component: the macromolecule, ions, and hydration water. Here, we demonstrate methods for robustly deconvoluting these signals, facilitating a more straightforward comparison with theory. Using SAXS data collected on an absolute intensity scale for short DNA duplexes in solution with Na(+), K(+), Rb(+), or Cs(+) counterions, we mathematically decompose the scattering profiles into components (DNA, water, and ions) and validate the decomposition using anomalous scattering measurements. In addition, we generate a library of physically motivated ion atmosphere models and rank them by agreement with the scattering data. The best-fit models have relatively compact ion atmospheres when compared to predictions from the mean-field Poisson-Boltzmann theory of electrostatics. Thus, the x-ray scattering methods presented here provide a valuable measurement of the global structure of the ion atmosphere that can be used to test electrostatics theories that go beyond the mean-field approximation.

SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell

“…Interestingly, the importance of ions, revealed by our comparative SAXS/SANS analysis, suggests a strategy to improve existing programs invoking explicit solvent (40)(41)(42)(43)(44)(45)75,76) by incorporating specific interactions of ions with charged surface residues. Further developments of SAXS/SANS programs are particularly crucial for biological macromolecular systems with complex and/or polyelectrolyte surfaces such as RNA/DNA molecules (27,(77)(78)(79), intrinsically disordered proteins (59,(80)(81)(82), and membrane protein/detergent complexes (83)(84)(85)) that all rely strongly on an accurate description of the HS.…”

Extracting water and ion distributions from solution x-ray scattering experiments

“…[14][15][16]18,34,35 Another approach uses heavy ion replacement, assuming the ion and water distributions are similar for the same type of ions (alkalies, for example). 14,20 By doing this, onlyF ion is allowed to vary while the co-ion and hydration terms are fixed. Subtracting the square roots of two measured intensities, therefore, gives the contribution from the counterion only,…”

“…Another technique is to use heavy ion replacement where instead one varies the ion identity to change the contrast. Using a novel analysis technique (instead of the simple subtraction between the two scattering curves as is done in conventional ASAXS), Meisburger et al 20 were able to separate the ion-DNA term from the water-DNA term and thus could gain insight into the nature of ion cloud around duplex DNA. The method assumes that both the ion and water distributions around DNA are not sensitive to ion type and it has applied successfully to alkali chlorides.…”

“…Meisburger et al express the total SAXS intensity as 20 I (q) = δ 2 solu P solu (q) + 2δ solu (δ ion N ion ) P solu−ion (q)…”

“…20 Both approaches, nonetheless, only show the solute-ion and ion-ion correlations in reciprocal space and are not readily extended to the water distribution.…”