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. 2016 Jun;8(2):139-149.
doi: 10.1007/s12551-016-0196-8.

Protein-DNA and ion-DNA interactions revealed through contrast variation SAXS

Joshua M Tokuda  1 , Suzette A Pabit  1 , Lois Pollack  1
Affiliations

Protein-DNA and ion-DNA interactions revealed through contrast variation SAXS

Joshua M Tokuda et al. Biophys Rev. 2016 Jun.

Abstract

Understanding how DNA carries out its biological roles requires knowledge of its interactions with biological partners. Since DNA is a polyanionic polymer, electrostatic interactions contribute significantly. These interactions are mediated by positively charged protein residues or charge compensating cations. Direct detection of these partners and/or their effect on DNA conformation poses challenges, especially for monitoring conformational dynamics in real time. Small-angle x-ray scattering (SAXS) is uniquely sensitive to both the conformation and local environment (i.e. protein partner and associated ions) of the DNA. The primary challenge of studying multi-component systems with SAXS lies in resolving how each component contributes to the measured scattering. Here, we review two contrast variation (CV) strategies that enable targeted studies of the structures of DNA or its associated partners. First, solution contrast variation enables measurement of DNA conformation within a protein-DNA complex by masking out the protein contribution to the scattering profile. We review a specific example, in which the real-time unwrapping of DNA from a nucleosome core particle is measured during salt-induced disassembly. The second method, heavy atom isomorphous replacement, reports the spatial distribution of the cation cloud around duplex DNA by exploiting changes in the scattering strength of cations with varying atomic numbers. We demonstrate the application of this approach to provide the spatial distribution of monovalent cations (Na+, K+, Rb+, Cs+) around a standard 25-base pair DNA. The CV strategies presented here are valuable tools for understanding DNA interactions with its biological partners.

Keywords: Contrast variation; DNA; Heavy atom isomorphous replacement; Ions; NCP; SAXS.

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Conflict of interest statement

Conflict of interest

Joshua M. Tokuda declares that he has no conflict of interest.

Suzette A. Pabit declares that she has no conflict of interest.

Lois Pollack declares that she has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Figures

Fig. 1
Fig. 1
Small-angle X-ray scattering (SAXS) is a solution-based approach that yields the low-resolution structures of macromolecules. Left A schematic of a typical SAXS experiment is shown. The sample is typically a buffered solution containing 2 mg/mL of protein, DNA, or protein–DNA complex. This sample oscillates through a quartz capillary to reduce radiation damage from the x-ray beam. The scattered x-rays are imaged onto an area detector while the primary beam is either blocked or greatly attenuated (as shown) by a beamstop. Right The images are pooled, averaged, and converted into profiles of intensity as a function of scattering vector, I(q), through azimuthal integration. For each sample, a corresponding measurement of the buffer alone is made and the resulting buffer profile is subtracted from the sample profile to obtain the macromolecular SAXS profile. SAXS intensities can be calibrated onto an absolute scale (in units of e2) through the measurement of water as a standard
Fig. 2
Fig. 2
Cartoon illustration of the principle behind solution contrast variation. a A color scale bar is shown with average electron density values for DNA, protein, and water. b, c Cartoon schematics of the nucleosome core particle (1AOI, Luger et al. 1997) in buffers with electron densities that vary according to the addition of 0 % (b) and 50 % (c) sucrose. The resulting contrasts (excess electron densities) are shown below each condition. d SAXS profiles for the DNA and histone proteins measured separately with and without sucrose. Note: in 50 % sucrose, the histone SAXS signal disappears, but the DNA is still visible due to its higher electron density
Fig. 3
Fig. 3
Application of solution contrast variation to monitor DNA unwrapping during the salt-induced disassembly of nucleosome core particles (NCP). a DNA models for the expected end states of the NCP at low [NaCl] (completely wrapped) and high [NaCl] (completely unwrapped). b P(R) curves for the NCP measured in 0 % sucrose and varied [NaCl]. c P(R) curves for the NCP measured in 50 % sucrose and varied [NaCl]. d P(R) curves determined for the models in (a). Peaks in the P(R) curves can be associated with structural features as follows: d 1, diameter of the duplex DNA; d 2, distance between overlapping DNA ends; d 3, diameter of the overall wrapped structure. These curves, specifically the difference between P(R) illustrate highlight how structural features emerge in the contrast matched condition
Fig. 4
Fig. 4
DNA models selected by ensemble optimization method (EOM) that best recapitulate the [NaCl]-dependent SAXS data for NCPs measured in 50 % sucrose. a Fit of the experimental data collected at 1.0 M NaCl with the theoretical SAXS curve generated from the ensemble of structures selected by EOM. Similar fits (not shown) were found for other [NaCl] concentrations. b Models representing the ensemble of structures selected by EOM that generate SAXS profiles that best fit the data measured at the indicated salt concentrations. χ 2 values assessing the overall fit to the experimental data and percentages reporting the weight of each model are shown. These models show how the DNA unwraps with increasing [NaCl]
Fig. 5
Fig. 5
Application of heavy atom isomorphous replacement to study the ion atmosphere around a 25-base pair DNA duplex. a Cartoon illustration of how increasing the atomic number of the monovalent cation cloud affects the scattering profile of the DNA–ion system. Ion size differences have been exaggerated to emphasize the increasing scattering factor. The dynamic spatial distributions of the different species of cations are assumed to be the same. b SAXS profiles for 50 μM DNA measured in 100 millimolar solutions of the monovalent chloride salts shown in (a). The increasing contrast for the heavier cations results in larger scattering signals. c The square root of the extrapolated forward scattering is shown to vary linearly with the effective ion contrast (see Eq. 5). This linearity is consistent with the assumption that the number and arrangement of these (excess) cations are identical (Meisburger et al. 2015)
Fig. 6
Fig. 6
Mathematical decomposition of the measured scattering of DNA duplexes and associated ions into contributions from partial structure factors. Shown here are the terms in Eq. 4 calculated for the DNA surrounded by a cloud of Rb+ ions. Details for this calculation are provided in Meisburger et al. (2015). Regularized fits to the data (black) were calculated using GNOM (Svergun 1992). Since DNA is always surrounded by a cloud of charge compensating ions, the mathematical decomposition illustrates the strong contributions of the DNA–ion cross term and the ion–ion scattering to the scattering profile of the DNA–ion system
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