From a structural average to the conformational ensemble of a DNA bulge

Abstract

Direct experimental measurements of conformational ensembles are critical for understanding macromolecular function, but traditional biophysical methods do not directly report the solution ensemble of a macromolecule. Small-angle X-ray scattering interferometry has the potential to overcome this limitation by providing the instantaneous distance distribution between pairs of gold-nanocrystal probes conjugated to a macromolecule in solution. Our X-ray interferometry experiments reveal an increasing bend angle of DNA duplexes with bulges of one, three, and five adenosine residues, consistent with previous FRET measurements, and further reveal an increasingly broad conformational ensemble with increasing bulge length. The distance distributions for the AAA bulge duplex (3A-DNA) with six different Au-Au pairs provide strong evidence against a simple elastic model in which fluctuations occur about a single conformational state. Instead, the measured distance distributions suggest a 3A-DNA ensemble with multiple conformational states predominantly across a region of conformational space with bend angles between 24 and 85 degrees and characteristic bend directions and helical twists and displacements. Additional X-ray interferometry experiments revealed perturbations to the ensemble from changes in ionic conditions and the bulge sequence, effects that can be understood in terms of electrostatic and stacking contributions to the ensemble and that demonstrate the sensitivity of X-ray interferometry. Combining X-ray interferometry ensemble data with molecular dynamics simulations gave atomic-level models of representative conformational states and of the molecular interactions that may shape the ensemble, and fluorescence measurements with 2-aminopurinesubstituted 3A-DNA provided initial tests of these atomistic models. More generally, X-ray interferometry will provide powerful benchmarks for testing and developing computational methods. helix-junction-helix | SAXSA grand challenge in biology is to understand the complex free-energy landscape of macromolecules and to decipher the resulting conformational ensembles. To perform their biological functions, macromolecules must adopt a multiplicity of conformations. Balancing and controlling different conformational states is central to biological processes including protein folding, allostery and signaling, and the stepwise assembly and function of macromolecular machines. To understand these complex molecules requires characterization of their free-energy landscapes-i.e., their equilibrium conformational ensembles. Precise measurements of conformational ensembles could allow quantitative modeling of the folding and function of biological macromolecules, would provide valuable experimental data to test current computational models and assumptions, and might facilitate the rational design of specifically acting inhibitors (1, 2).Techniques including NMR and EPR relaxation have been developed to incisively probe motions in the ensemble on different time scales, ranging from pic...

From static to dynamic: the need for structural ensembles and a predictive model of RNA folding and function

“…NMR RDC approaches have proven powerful for probing rotational degrees of freedom, and, in principle, complementary information can be obtained from X-ray scattering interference (XSI), an approach developed by Harbury and colleagues that employs two site-specifically attached Au-nanocrystals and provides an instantaneous distance-probability distribution via the scattering interference pattern of these nanocrystals [35 • ,36 • ,37 • • , Shi et al in press]. Given the large number of sequence combinations in need of exploration, developing information-rich, high-throughput biophysical assays is a key challenge.…”

Characterizing the bending and flexibility induced by bulges in DNA duplexes

“…A recent study of bulged duplexes in DNA observed an increase in flexibility of the motifs (a wider range of bend angles was observed) upon an increase in bulge loop size, findings which are broadly consistent with the predictions of oxDNA for the average bend angle distributions. 67 Interestingly, Bailor and co-workers 21,39 have reported results for RNA where the bend angle in bulged RNA duplexes monotonically increased up to about M = 7 and then plateaued. This is again similar to the behavior we see for the oxDNA bend angles for the unstacked configurations, which rises and then plateaus at M = 8.…”

Thermodynamic examination of 1- to 5-nt purine bulge loops in RNA and DNA constructs

“…NMR has been used to study the dynamics of trinucleotide bulge loop structures (Zhang et al 2006(Zhang et al , 2010. X-ray interferometry studies have provided information on the ensemble of bent conformations present in trinucleotide DNA bulge loop constructs (Shi et al 2014).…”