Experimental approaches for investigating ion atmospheres around nucleic acids and proteins

Abstract

Ionic interactions are crucial to biological functions of DNA, RNA, and proteins. Experimental research on how ions behave around biological macromolecules has lagged behind corresponding theoretical and computational research. In the 21st century, quantitative experimental approaches for investigating ionic interactions of biomolecules have become available and greatly facilitated examinations of theoretical electrostatic models. These approaches utilize anomalous small-angle X-ray scattering, atomic emission spectroscopy, mass spectrometry, or nuclear magnetic resonance (NMR) spectroscopy. We provide an overview on the experimental methodologies that can quantify and characterize ions within the ion atmospheres around nucleic acids, proteins, and their complexes.

Keywords: AES, atomic emission spectroscopy; ASAXS, anomalous small-angle X-ray scattering; BE, buffer equilibration; Dynamics; Electrostatic interactions; ICP-MS, inductively coupled plasma mass spectrometry; Ion atmosphere; Ionic diffusion; NMR, nuclear magnetic resonance; PRE, paramagnetic relaxation enhancement; SAXS, small-angle X-ray scattering; Spatial distribution.

Graphical abstract

Fig. 1

Concepts of the ion atmosphere and relevant theories. (A) Charge neutralization for the space comprising of a macromolecule and its ion atmosphere. (B) View of the counterion condensation theory. λ_B is the Bjerrum length. For nucleic acids, ΔN_counterion = ΔN_cation and ΔN_coion = ΔN_anion. (C) View of the Poisson-Boltzmann theory. Symbols used in the ion-excess numbers ΔN_cation and ΔN_anion are as follows: n_A is Avogadro’s number; c is the bulk ion concentration in mol/L units; ρ defines ion accessibility in the box with ρ = 1 for accessible regions and ρ = 0 for regions that are inaccessible due to macromolecular atoms; q, the ionic charge; e, the elementary charge; k_B, the Boltzmann constant; T, the temperature; and v, the volume in m³. A factor of 1000 is for the conversion of the volume unit from L to m³. Subtraction of 1 in the integral corresponds to subtraction of the Boltzmann factor for the background with an electrostatic potential of zero. The shown equations for ion excess are for monovalent ions.

Fig. 2

Ion-counting methods for quantifying ions in the ion atmosphere. The ion excess per macromolecule (ΔN_ion) is determined from the ion concentrations in the final solution (c_ion,sol) and in the reference buffer used for the equilibration (c_ion,ref) as well as the macromolecular concentration in the final solution (c_mac,sol).

Fig. 3

NMR-based quantification of anions accumulated around the Antp homeodomain, BPTI, and ubiquitin. Note that the measured ΔN_anion was smaller than the overall charge valence Z. This means that the charge neutralization occurs not only via accumulation of anions, but also via exclusion of cations from the ion atmosphere. Adopted from Yu et al.

Fig. 4

NMR paramagnetic relaxation enhancement (PRE)-based approach for investigating the spatial distribution of anions around the Antp homeodomain and its complex with 15-bp DNA. The data were adopted from Yu et al. Comparison of PRE arising from analogous anionic and neutral paramagnetic cosolutes provides site-specific information about ion accumulation or exclusion. The data shown on the left-hand side suggest that anions are accumulated around the Antp homeodomain in the free state but excluded upon formation of the protein-DNA complex.