Recording and Analyzing Nucleic Acid Distance Distributions with X-Ray Scattering Interferometry (XSI).

Most structural techniques provide averaged information or information about a single predominant conformational state. However, biological macromolecules typically function through series of conformations. Therefore, a complete understanding of macromolecular structures requires knowledge of the ensembles that represent probabilities on a conformational free energy landscape. Here we describe an emerging approach, X-ray scattering interferometry (XSI), a method that provides instantaneous distance distributions for molecules in solution. XSI uses gold nanocrystal labels site-specifically attached to a macromolecule and measures the scattering interference from pairs of heavy metal labels. The recorded signal can directly be transformed into a distance distribution between the two probes. We describe the underlying concepts, present a detailed protocol for preparing samples and recording XSI data, and provide a custom-written graphical user interface to facilitate XSI data analysis. © 2018 by John Wiley & Sons, Inc.

Gold nanocrystal labels provide a sequence-to-3D structure map in SAXS reconstructions.

Small-angle x-ray scattering (SAXS) is a powerful technique to probe the structure of biological macromolecules and their complexes under virtually arbitrary solution conditions, without the need for crystallization. While it is possible to reconstruct molecular shapes from SAXS data ab initio, the resulting electron density maps have a resolution of ~1 nm and are often insufficient to reliably assign secondary structure elements or domains. We show that SAXS data of gold-labeled samples significantly enhance the information content of SAXS measurements, allowing the unambiguous assignment of macromolecular sequence motifs to specific locations within a SAXS structure. We first demonstrate our approach for site-specifically internally and end-labeled DNA and an RNA motif. In addition, we present a protocol for highly uniform and site-specific labeling of proteins with small (~1.4 nm diameter) gold particles and apply our method to the signaling protein calmodulin. In all cases, the position of the small gold probes can be reliably identified in low-resolution electron density maps. Enhancing low-resolution measurements by site-selective gold labeling provides an attractive approach to aid modeling of a large range of macromolecular systems.

Absolute Intramolecular Distance Measurements with Angstrom-Resolution Using Anomalous Small-Angle X-ray Scattering.

Accurate determination of molecular distances is fundamental to understanding the structure, dynamics, and conformational ensembles of biological macromolecules. Here we present a method to determine the full distance distribution between small (∼7 Å radius) gold labels attached to macromolecules with very high-precision (≤1 Å) and on an absolute distance scale. Our method uses anomalous small-angle X-ray scattering close to a gold absorption edge to separate the gold-gold interference pattern from other scattering contributions. Results for 10-30 bp DNA constructs achieve excellent signal-to-noise and are in good agreement with previous results obtained by single-energy SAXS measurements without requiring the preparation and measurement of single labeled and unlabeled samples. The use of small gold labels in combination with ASAXS read out provides an attractive approach to determining molecular distance distributions that will be applicable to a broad range of macromolecular systems.

Structural ensemble and microscopic elasticity of freely diffusing DNA by direct measurement of fluctuations.

Precisely measuring the ensemble of conformers that a macromolecule populates in solution is highly challenging. Thus, it has been difficult to confirm or falsify the predictions of nanometer-scale dynamical modeling. Here, we apply an X-ray interferometry technique to probe the solution structure and fluctuations of B-form DNA on a length scale comparable to a protein-binding site. We determine an extensive set of intrahelix distance distributions between pairs of probes placed at distinct points on the surface of the DNA duplex. The distributions of measured distances reveal the nature and extent of the thermally driven mechanical deformations of the helix. We describe these deformations in terms of elastic constants, as is common for DNA and other polymers. The average solution structure and microscopic elasticity measured by X-ray interferometry are in striking agreement with values derived from DNA-protein crystal structures and measured by force spectroscopy, with one exception. The observed microscopic torsional rigidity of DNA is much lower than is measured by single-molecule twisting experiments, suggesting that torsional rigidity increases when DNA is stretched. Looking forward, molecular-level interferometry can provide a general tool for characterizing solution-phase structural ensembles.

Expedient synthesis of a modular phosphate affinity reagent.

Isolation and identification of phosphorylated macromolecules is essential for the deconvolution of most biological regulatory networks. Koike and co-workers recently reported the application of a dinuclear zinc-(pyridylmethyl)amine complex to phosphate-specific affinity purifications and gave it the shorthand name "phos-tag". This complex is valuable for studying phosphorylation because it binds selectively to phosphate dianion in the presence of acidic functional groups at physiological pH, and because the binding is largely independent of molecular context. These properties of phos-tag recommend it for applications in phosphoproteomics, metabolomics, and nucleic acid biology. The catch has been that the molecule is difficult to make and prohibitively expensive to buy. Here, we describe an efficient and inexpensive synthesis of a phos-tag derivative with a versatile alkyne handle. The alkyne handle allows for attachment of phos-tag to alkyl azides via the copper(I)-catalyzed azide-alkyne cycloaddition reaction ("click chemistry"). We characterize the phosphate binding behavior of the new phos-tag derivative in a variety of experimental assays, including its conjugation to a fluorescent reporter, to acrylamide gels, and to sepharose chromatography resin. The synthesis we report should enable a broader use of phos-tag for phosphate-related biochemistry, as both an analytical and a preparative reagent.

Remeasuring the double helix.

DNA is thought to behave as a stiff elastic rod with respect to the ubiquitous mechanical deformations inherent to its biology. To test this model at short DNA lengths, we measured the mean and variance of end-to-end length for a series of DNA double helices in solution, using small-angle x-ray scattering interference between gold nanocrystal labels. In the absence of applied tension, DNA is at least one order of magnitude softer than measured by single-molecule stretching experiments. Further, the data rule out the conventional elastic rod model. The variance in end-to-end length follows a quadratic dependence on the number of base pairs rather than the expected linear dependence, indicating that DNA stretching is cooperative over more than two turns of the DNA double helix. Our observations support the idea of long-range allosteric communication through DNA structure.

A molecular ruler for measuring quantitative distance distributions.

We report a novel molecular ruler for measurement of distances and distance distributions with accurate external calibration. Using solution X-ray scattering we determine the scattering interference between two gold nanocrystal probes attached site-specifically to a macromolecule of interest. Fourier transformation of the interference pattern provides a model-independent probability distribution for the distances between the probe centers-of-mass. To test the approach, we measure end-to-end distances for a variety of DNA structures. We demonstrate that measurements with independently prepared samples and using different X-ray sources are highly reproducible, we demonstrate the quantitative accuracy of the first and second moments of the distance distributions, and we demonstrate that the technique recovers complex distribution shapes. Distances measured with the solution scattering-interference ruler match the corresponding crystallographic values, but differ from distances measured previously with alternate ruler techniques. The X-ray scattering interference ruler should be a powerful tool for relating crystal structures to solution structures and for studying molecular fluctuations.