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Disordered RNA chaperones can enhance nucleic acid folding via local charge screening - PubMed

  • ️Tue Jan 01 2019

Disordered RNA chaperones can enhance nucleic acid folding via local charge screening

Erik D Holmstrom et al. Nat Commun. 2019.

Abstract

RNA chaperones are proteins that aid in the folding of nucleic acids, but remarkably, many of these proteins are intrinsically disordered. How can these proteins function without a well-defined three-dimensional structure? Here, we address this question by studying the hepatitis C virus core protein, a chaperone that promotes viral genome dimerization. Using single-molecule fluorescence spectroscopy, we find that this positively charged disordered protein facilitates the formation of compact nucleic acid conformations by acting as a flexible macromolecular counterion that locally screens repulsive electrostatic interactions with an efficiency equivalent to molar salt concentrations. The resulting compaction can bias unfolded nucleic acids towards folding, resulting in faster folding kinetics. This potentially widespread mechanism is supported by molecular simulations that rationalize the experimental findings by describing the chaperone as an unstructured polyelectrolyte.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1

Nucleic-acid chaperone binds hairpin with high affinity. a Molecular representations of the folded and unfolded DNA hairpin with the phosphate, sugar, and base moieties colored in red, pink, and white, respectively; magenta and green spheres indicate the acceptor and donor dyes within the 5′-3′ FRET-labeled hairpin. The positively charged and intrinsically disordered nucleocapsid domain of the hepatitis C virus core protein (NCD, cyan) chaperones hairpin formation. b Transfer efficiency histograms from measurements of labeled hairpin at increasing concentrations of unlabeled chaperone under conditions with near-physiological concentrations of monovalent cations (~75 mM from phosphate buffer (PB) and 80 mM from NaCl). c Mean transfer efficiencies, 〈E〉, from b plotted against chaperone (NCD) concentration. Source data for mean values are provided as a Source Data file. Apparent dissociation constants, appKd, are determined from fits to a 2-state binding isotherm (see Methods section). d Double-logarithmic dependence of appKd on sodium ion concentration with fit to a power-law of the form fx=axn (solid line, 99% confidence band in gray), where x is the concentration of sodium ions and n is related to the number of counterions released when NCD binds to the hairpin

Fig. 2
Fig. 2

Chaperone binding accelerates hairpin folding. a Representative donor (green) and acceptor (magenta) fluorescence time traces depicting unassisted folding of the surface-immobilized 5′-3′ FRET-labeled hairpin. The uncorrected transfer efficiency, Ê, (black) and the most likely state trajectory (gray) based on the Viterbi algorithm are shown below. b Analogous to a, but depicting chaperone-assisted hairpin folding with saturating concentrations of NCD (150 nM) to ensure that hairpin molecules are almost always chaperone-associated. c Survival probability plots of folded- (upper left) and unfolded-state (lower left) dwell times in the absence of chaperone, fit with single-exponential decays to determine the unfolding and folding rate constants, kU and kF, respectively; 〈kU〉 and 〈kF〉 represent mean values obtained from 54 single-molecule measurements. The donor-acceptor fluorescence cross-correlation function (right) decorrelates with a rate constant that is consistent with the sum of kU and kF from the dwell-time analysis. d Analogous to c, but for chaperone-assisted folding; 〈kU〉 and 〈kF〉 represent mean values obtained from 26 single-molecule measurements. The black lines in c and d are fits with single exponentials with 99% confidence bands shown in gray. e Representative fluorescence time trace measured at a chaperone concentration near appKd. Transitions between chaperone-bound (cyan shading) and chaperone-free (red shading) kinetic regimes arise from binding and dissociation of NCD. f Kinetic 4-state model for chaperone-assisted folding. The folded, F, and unfolded, U, conformations of the hairpin freely interconvert in the absence of chaperone, C, with an equilibrium constant that favors U. When the chaperone is bound, the unfolded, UC, and folded, FC, conformations of the hairpin still interconvert, but with an equilibrium constant that favors F. g Trajectory from coarse-grained molecular dynamics simulation showing binding (cyan shading) and dissociation (red shading) of the chaperone to the hairpin concomitant with the formation of intermolecular contacts (gray). The folding and unfolding of the hairpin, monitored via native contacts (black), is shifted to favor hairpin formation when the chaperone is bound. Structural representations are taken from the simulations (see Supplementary Movies 1 and 2). Source data for mean values in c and d are provided as a Source Data File

Fig. 3
Fig. 3

Structural properties of chaperone-hairpin complex. a Molecular surface representations of the binding partners colored by electrostatic potential (red, negative; blue, positive). Arrows indicate the approximate location of labeling sites. b Donor-donor (light green), acceptor-acceptor (magenta), and donor-acceptor (gray) fluorescence correlation functions for FRET-labeled NCD S2C-T65C in isolation (top), bound to the hairpin (middle), and bound to the folding-incompetent hairpin (bottom). Solid black lines are global fits of the three respective correlation functions, which are used to determine the reconfiguration times, τr, of the chaperone. c Mean transfer efficiencies for all labeling pairs from the mapping experiment (triangles) at 30 mM PB pH 7.0 and the coarse-grained molecular simulations (circles). Mean values are provided in the Source Data file. Labeling positions in the chaperone and the hairpin are shown in cyan and red, respectively. The uncertainties associated with transfer efficiency values are ~0.01 based on multiple simulations and ~0.03 for the experiments (error dominated by instrument calibration). d Molecular representations of the disordered nucleic acid-chaperone complex from the simulations (see Supplementary Movies 1 and 2); color code as in Fig. 1a. e Correlation between experimental and simulated mean transfer efficiencies. The dashed gray line is the identity line, the solid black line is a linear regression

Fig. 4
Fig. 4

NCD functions as a macromolecular counterion. Transfer efficiency histograms for a salt-assisted and b chaperone-assisted folding of the 5′-3′ FRET-labeled DNA hairpin. Increasing concentrations of either NaCl or NCD promote formation of the hairpin, but the transition midpoint concentrations (i.e., appKd and [NaCl]1/2) for the two species differ by a factor of ∼107. c Mean inter-dye distance for the folding-incompetent hairpin as a function of sodium ion concentration. Source data for mean values are provided as a Source Data file. The compaction of the unfolded nucleic acid induced by chaperone binding (cyan arrow) is comparable to that present at molar concentrations of NaCl. The dashed black line results from a fit to a worm-like chain with a salt-dependent (i.e., electrostatic) persistence length (see Methods section). d Mean folding rate constant for hairpin formation as a function of NaCl concentration. Source data for mean values are provided as a Source Data file. Chaperone binding gives rise to a folding rate constant comparable to solutions containing molar concentrations of NaCl (cyan arrow). The extrapolation (dashed black line) is a fit of the data to a power law of the form fx=axn, where x is the concentration of NaCl, and n is related to the number of counterions taken up upon forming the transition state

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