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[Preprint]. 2024 Sep 8:2024.09.05.611539.
doi: 10.1101/2024.09.05.611539.

Early Events in G-quadruplex Folding Captured by Time-Resolved Small-Angle X-Ray Scattering

Robert C Monsen  1 , T Michael Sabo  1 , Robert Gray  1 , Jesse B Hopkins  2 , Jonathan B Chaires  1
Affiliations

Early Events in G-quadruplex Folding Captured by Time-Resolved Small-Angle X-Ray Scattering

Robert C Monsen et al. bioRxiv. .

Update in

Abstract

Time-resolved small-angle X-ray experiments (TR-SAXS) are reported here that capture and quantify a previously unknown rapid collapse of the unfolded oligonucleotide as an early step in G4 folding of hybrid 1 and hybrid 2 telomeric G-quadruplex structures. The rapid collapse, initiated by a pH jump, is characterized by an exponential decrease in the radius of gyration from 20.6 to 12.6 Å. The collapse is monophasic and is complete in less than 600 ms. Additional hand-mixing pH-jump kinetic studies show that slower kinetic steps follow the collapse. The folded and unfolded states at equilibrium were further characterized by SAXS studies and other biophysical tools, to show that G4 unfolding was complete at alkaline pH, but not in LiCl solution as is often claimed. The SAXS Ensemble Optimization Method (EOM) analysis reveals models of the unfolded state as a dynamic ensemble of flexible oligonucleotide chains with a variety of transient hairpin structures. These results suggest a G4 folding pathway in which a rapid collapse, analogous to molten globule formation seen in proteins, is followed by a confined conformational search within the collapsed particle to form the native contacts ultimately found in the stable folded form.

Keywords: G-quadruplex; Telomere folding; Time-resolved SAXS.

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Figures

Figure 1.
Figure 1.
(A) CD spectra of 2GKU in KCL (black), LiCl (blue), KCl at pH 11.5 (purple) and KCl at 98 °C (red). (B) Sedimentation coefficient distributions for 2GKU in KCl (black), LiCl (blue) and KCl at pH 11.5 (purple). The sedimentation of an unstructured single-stranded reference oligonucleotide, T24, is shown in green.
Figure 2.
Figure 2.
Equilibrium SAXS analysis of 2GKU. (A) Scattering profiles, (B) Dimensionless (re-binned) Kratky plots, and (C) Normalized P(r) distributions of 2GKU in KCl at pH 7.2 (black), LiCl at pH 7.2 (red), and KCl at pH 11.5 (blue).
Figure 3.
Figure 3.
EOM analysis results for 2GKU. (A-B) EOM distributions for the radius of gyration (A) and Dmax (B) for the total pool of conformers (black) and the selected ensemble of flexible structures (red). (C) pH 11.5 scattering curve with EOM fit overlaid in red and residuals below. (D) Best fit ensemble of conformers chosen by EOM from duplicate 500 ns implicitly solvated MD simulations starting from single-stranded 2GKU showing the most extended (top) to the most compact conformations (bottom) oriented with 5’ end on the left. Calculated Dmax values and % ensemble weight are given above each model. EOM statistics: Rflex (random) / Rsigma: ~ 84.25% (~ 90.67%) / 0.85.
Figure 4.
Figure 4.
2GKU pH-jump spectra and kinetics. Folding was initiated by manually mixing a small volume of concentrated hydrochloric acid into the alkaline (pH 11.5) buffer solution to drop it to neutral pH (final pH = 7.1). (A) CD spectra of the alkaline denatured 2GKU pre- and post- pH-jump monitored over 11,000 s. (B) Fractional component plot from SVD with relaxation times overlaid based on single exponential fits. (C) Deconvoluted CD spectra for each component in B from SVD. (D) Residuals from the nonlinear least-squares fit to the data from SVD analysis.
Figure 5.
Figure 5.
Time-resolved SAXS results of 2GKU following pH jump from 11.5 to 7.2. (A) Selected normalized P(r) distributions of the pH-induced structural collapse of 2GKU bracketed by the equilibrium SAXS profiles showing the conversion from an extended unstructured species to a globular and compact particle of nearly identical size and shape as the folded hybrid 1 form. (B) Dimensionless Kratky plots (data re-binned for clarity with log mode and re-bin factor 4) showing the transition from the denatured flexible chain to a compact globular form that is nearly identical to the equilibrium 2GKU scattering. (C) REGALS derived regularized P(r) distributions comparing the two deconvoluted components (solid lines) with the equilibrium SAXS 2GKU distributions (dashed lines). (D) Regularized component concentration profiles from REGALS deconvolution with single exponential decay relaxation times overlaid and fits shown in blue. Red and black curves correspond to the solid red and black P(r) distributions in C.
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