2,853 research outputs found
Mechanical unfolding of RNA: From hairpins to structures with internal multiloops
Mechanical unfolding of RNA structures, ranging from hairpins to ribozymes,
using laser optical tweezer (LOT) experiments have begun to reveal the features
of the energy landscape that cannot be easily explored using conventional
experiments. Upon application of constant force (), RNA hairpins undergo
cooperative transitions from folded to unfolded states whereas subdomains of
ribozymes unravel one at a time. Here, we use a self-organized polymer (SOP)
model and Brownian dynamics simulations to probe mechanical unfolding at
constant force and constant-loading rate of four RNA structures of varying
complexity. Our work shows (i) the response of RNA to force is largely
determined by the native structure; (ii) only by probing mechanical unfolding
over a wide range of forces can the underlying energy landscape be fully
explored.Comment: 26 pages, 6 figures, Biophys. J. (in press
Folding and unfolding of a triple-branch DNA molecule with four conformational states
Single-molecule experiments provide new insights into biological processes
hitherto not accessible by measurements performed on bulk systems. We report on
a study of the kinetics of a triple-branch DNA molecule with four
conformational states by pulling experiments with optical tweezers and
theoretical modelling. Three distinct force rips associated with different
transitions between the conformational states are observed in the folding and
unfolding trajectories. By applying transition rate theory to a free energy
model of the molecule, probability distributions for the first rupture forces
of the different transitions are calculated. Good agreement of the theoretical
predictions with the experimental findings is achieved. Furthermore, due to our
specific design of the molecule, we found a useful method to identify
permanently frayed molecules by estimating the number of opened basepairs from
the measured force jump values.Comment: 17 pages, 12 figure
Refolding dynamics of stretched biopolymers upon force quench
Single molecule force spectroscopy methods can be used to generate folding
trajectories of biopolymers from arbitrary regions of the folding landscape. We
illustrate the complexity of the folding kinetics and generic aspects of the
collapse of RNA and proteins upon force quench, using simulations of an RNA
hairpin and theory based on the de Gennes model for homopolymer collapse. The
folding time, , depends asymmetrically on and
where () is the stretch (quench) force, and
is the transition mid-force of the RNA hairpin. In accord with
experiments, the relaxation kinetics of the molecular extension, , occurs
in three stages: a rapid initial decrease in the extension is followed by a
plateau, and finally an abrupt reduction in that occurs as the native
state is approached.
The duration of the plateau increases as decreases
(where is the time in which the force is reduced from to ).
Variations in the mechanisms of force quench relaxation as is altered
are reflected in the experimentally measurable time-dependent entropy, which is
computed directly from the folding trajectories. An analytical solution of the
de Gennes model under tension reproduces the multistage stage kinetics in
. The prediction that the initial stages of collapse should also be a
generic feature of polymers is validated by simulation of the kinetics of
toroid (globule) formation in semiflexible (flexible) homopolymers in poor
solvents upon quenching the force from a fully stretched state. Our findings
give a unified explanation for multiple disparate experimental observations of
protein folding.Comment: 31 pages 11 figure
Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot
The functions of RNA pseudoknots (PKs), which are minimal tertiary structural
motifs and an integral part of several ribozymes and ribonucleoprotein
complexes, are determined by their structure, stability and dynamics.
Therefore, it is important to elucidate the general principles governing their
thermodynamics/folding mechanisms. Here, we combine experiments and simulations
to examine the folding/unfolding pathways of the VPK pseudoknot, a variant of
the Mouse Mammary Tumor Virus (MMTV) PK involved in ribosomal frameshifting.
Fluorescent nucleotide analogs (2-aminopurine and pyrrolocytidine) placed at
different stem/loop positions in the PK, and laser temperature-jump approaches
serve as local probes allowing us to monitor the order of assembly of VPK with
two helices with different intrinsic stabilities. The experiments and molecular
simulations show that at 50 mM KCl the dominant folding pathway populates only
the more stable partially folded hairpin. As the salt concentration is
increased a parallel folding pathway emerges, involving the less stable hairpin
structure as an alternate intermediate. Notably, the flux between the pathways
is modulated by the ionic strength. The findings support the principle that the
order of PK structure formation is determined by the relative stabilities of
the hairpins, which can be altered by sequence variations or salt
concentrations. Our study not only unambiguously demonstrates that PK folds by
parallel pathways, but also establishes that quantitative description of RNA
self-assembly requires a synergistic combination of experiments and
simulations.Comment: Supporting Information include
Forced-unfolding and force-quench refolding of RNA hairpins
Using coarse-grained model we have explored forced-unfolding of RNA hairpin
as a function of and the loading rate (). The simulations and
theoretical analysis have been done without and with the handles that are
explicitly modeled by semiflexible polymer chains. The mechanisms and time
scales for denaturation by temperature jump and mechanical unfolding are vastly
different. The directed perturbation of the native state by results in a
sequential unfolding of the hairpin starting from their ends whereas thermal
denaturation occurs stochastically. From the dependence of the unfolding rates
on and we show that the position of the unfolding transition state
(TS) is not a constant but moves dramatically as either or is
changed. The TS movements are interpreted by adopting the Hammond postulate for
forced-unfolding. Forced-unfolding simulations of RNA, with handles attached to
the two ends, show that the value of the unfolding force increases (especially
at high pulling speeds) as the length of the handles increases. The pathways
for refolding of RNA from stretched initial conformation, upon quenching
to the quench force , are highly heterogeneous. The refolding times, upon
force quench, are at least an order of magnitude greater than those obtained by
temperature quench. The long -dependent refolding times starting from
fully stretched states are analyzed using a model that accounts for the
microscopic steps in the rate limiting step which involves the trans to gauche
transitions of the dihedral angles in the GAAA tetraloop. The simulations with
explicit molecular model for the handles show that the dynamics of force-quench
refolding is strongly dependent on the interplay of their contour length and
the persistence length, and the RNA persistence length.Comment: 42 pages, 15 figures, Biophys. J. (in press
Force-induced misfolding in RNA
RNA folding is a kinetic process governed by the competition of a large
number of structures stabilized by the transient formation of base pairs that
may induce complex folding pathways and the formation of misfolded structures.
Despite of its importance in modern biophysics, the current understanding of
RNA folding kinetics is limited by the complex interplay between the weak
base-pair interactions that stabilize the native structure and the disordering
effect of thermal forces. The possibility of mechanically pulling individual
molecules offers a new perspective to understand the folding of nucleic acids.
Here we investigate the folding and misfolding mechanism in RNA secondary
structures pulled by mechanical forces. We introduce a model based on the
identification of the minimal set of structures that reproduce the patterns of
force-extension curves obtained in single molecule experiments. The model
requires only two fitting parameters: the attempt frequency at the level of
individual base pairs and a parameter associated to a free energy correction
that accounts for the configurational entropy of an exponentially large number
of neglected secondary structures. We apply the model to interpret results
recently obtained in pulling experiments in the three-helix junction S15 RNA
molecule (RNAS15). We show that RNAS15 undergoes force-induced misfolding where
force favors the formation of a stable non-native hairpin. The model reproduces
the pattern of unfolding and refolding force-extension curves, the distribution
of breakage forces and the misfolding probability obtained in the experiments.Comment: 28 pages, 11 figure
Structural ultrafast dynamics of macromolecules: diffraction of free DNA and effect of hydration
Of special interest in molecular biology is the study of structural and conformational changes which are free of the additional effects of the environment. In the present contribution, we report on the ultrafast unfolding dynamics of a large DNA macromolecular ensemble in vacuo for a number of temperature jumps, and make a comparison with the unfolding dynamics of the DNA in aqueous solution. A number of coarse-graining approaches, such as kinetic intermediate structure (KIS) model and ensemble-averaged radial distribution functions, are used to account for the transitional dynamics of the DNA without sacrificing the structural resolution. The studied ensembles of DNA macromolecules were generated using distributed molecular dynamics (MD) simulations, and the ensemble convergence was ensured by monitoring the ensemble-averaged radial distribution functions and KIS unfolding trajectories. Because the order–disorder transition in free DNA implies unzipping, coiling, and strand-separation processes which occur consecutively or competitively depending on the initial and final temperature of the ensemble, DNA order–disorder transition in vacuo cannot be described as a two-state (un)folding process
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