Characterizing the conformational free-energy landscape of RNA using single-molecule field-effect transistors

We have developed and used high-time-resolution, single-molecule field-effect transistors (smFETs) to characterize the con-formational free-energy landscape of RNA stem-loops. Stem-loops are some of the most common RNA structural motifs and serve as building blocks for the formation of more complex RNA structures. Given their prevalence and integral role in RNA folding, the kinetics of stem-loop (un)folding has been extensively characterized using both experimental and computational approaches. Interestingly, these studies have reported vastly disparate timescales of (un)folding, which has been recently in-terpreted as evidence that (un)folding of even simple stem-loops occurs on a highly rugged conformational energy landscape. Because smFETs do not rely on fluorophore reporters of conformation or on the application of mechanical (un)folding forces, they provide a unique and complementary approach that has allowed us to directly monitor tens of thousands of (un)folding events of individual stem-loops at a 200 μs time resolution. Our results show that under our experimental conditions, stem-loops fold and unfold over a 1-200 ms timescale during which they transition between ensembles of unfolded and folded conformations, the latter of which is composed of at least two sub-populations. The 1-200 ms timescale of (un)folding we observe here indicates that smFETs report on complete (un)folding trajectories in which relatively extended unfolded con-formations of the RNA spend long periods of time wandering the free-energy landscape before sampling one of several mis-folded conformations or, alternatively, the natively folded conformation. Our findings demonstrate how the combination of single-molecule sensitivity and high time resolution makes smFETs unique and powerful tools for characterizing the con-formational free-energy landscape of RNA and highlight the extremely rugged landscape on which even the simplest RNA structural elements fold.

Dissociation pathways of the p53 DNA binding domain from DNA and critical roles of key residues elucidated by dPaCS-MD/MSM

The dissociation process of the DNA binding domain of p53 (p53-DBD) from a DNA duplex that contains the consensus sequence, which is the specific target of p53-DBD, was investigated by a combination of dissociation parallel cascade selection molecular dynamics (dPaCS-MD) and the Markov state model (MSM). Based on an all-atom model including explicit solvent, we first simulated the p53-DBD dissociation processes by 75 trials of dPaCS-MD, which required an average simulation time of 11.2 ± 2.2 ns per trial. By setting the axis of the DNA duplex as the Z-axis and the binding side of p53-DBD on DNA as the + side of the X-axis, we found that dissociations took place along the +X and −Y directions (−Y directions) in 93% of the cases, while 7% of the cases moved along +X and +Y directions (+Y directions). Toward the −Y directions, p53-DBD dissociated first from the major groove and then detached from the minor groove, while unbinding from the minor groove occurred first in dissociations along the +Y directions. Analysis of the free energy landscape by MSM showed that loss of the minor groove interaction with p53-DBD toward the +Y directions incurred a relatively high energy cost (1.1 kcal/mol) upon a critical transition, whereas major groove detachment more frequently occurred with lower free energy costs. The standard binding free energy calculated from the free energy landscape was −10.9 ± 0.4 kcal/mol, which agrees with an experimental value of –11.1 kcal/mol. These results indicate that the dPaCS-MD/MSM combination can be a powerful tool to investigate dissociation mechanisms of two large molecules. Minor groove binding is mainly stabilized by R248, identified as the most important residue that tightly binds deep inside the minor groove. Analysis of the p53 key residues for DNA binding indicates high correlations with cancer-related mutations, confirming that impairment of the interactions between p53-DBD and DNA can be frequently related to cancer.

Free energy landscape and kinetics of phase transition in two coupled SYK models and the corresponding wormhole-two black hole switching

We propose that the thermodynamics and the kinetics of the phase transition between wormhole and two black hole described by the two coupled SYK model can be investigated in terms of the stochastic dynamics on the underlying free energy landscape. We assume that the phase transition is a stochastic process under the thermal fluctuations. By quantifying the underlying free energy landscape, we study the phase diagram, the kinetic time and its fluctuations in details, which reveal the underlying thermodynamics and kinetics. It is shown that the first order phase transition between wormhole and two black hole described by two coupled SYK model is analogous to the Van der Waals phase transition. Therefore, the emergence of wormhole and two black hole phases, the phase transition and associated kinetics can be quantitatively addressed in our free energy landscape and kinetic framework through the dependence on the barrier height and the temperature.

A Free-Energy Landscape Analysis of Calmodulin Obtained from an NMR Data-Utilized Multi-Scale Divide-and-Conquer Molecular Dynamics Simulation

Calmodulin (CaM) is a multifunctional calcium-binding protein, which regulates a variety of biochemical processes. CaM acts through its conformational changes and complex formation with its target enzymes. CaM consists of two globular domains (N-lobe and C-lobe) linked by an extended linker region. Upon calcium binding, the N-lobe and C-lobe undergo local conformational changes, followed by a major conformational change of the entire CaM to wrap the target enzyme. However, the regulation mechanisms, such as allosteric interactions, which regulate the large structural changes, are still unclear. In order to investigate the series of structural changes, the free-energy landscape of CaM was obtained by multi-scale divide-and-conquer molecular dynamics (MSDC-MD). The resultant free-energy landscape (FEL) shows that the Ca2+ bound CaM (holo-CaM) would take an experimentally famous elongated structure, which can be formed in the early stage of structural change, by breaking the inter-domain interactions. The FEL also shows that important interactions complete the structural change from the elongated structure to the ring-like structure. In addition, the FEL might give a guiding principle to predict mutational sites in CaM. In this study, it was demonstrated that the movement process of macroscopic variables on the FEL may be diffusive to some extent, and then, the MSDC-MD is suitable to the parallel computation.

The Ageing and Rejuvenation of Soft, Glassy Complex Fluids

<p>Suspensions of multiarm star polymers are studied as models for soft colloidal interactionsin colloidal glasses. Establishing a pre-shearing protocol which ensures a reproducible initial state (the "rejuvenation" of the system), we report here the stress evolution from startup for two different concentrations for a range of shear rates using conventional rheological techniques. We show the existence of critical shear rateswhich are functions of the concentration. When the suspensions are sheared at rates below the critical rates, the stress rises to a common value which is also a function of the concentration. The system thus evolves into a yield stress-like fluid. This behavior manifests itself as an evolution from a monotonic, slightly shear-thinning flow curve to a flow curve dominated by a stress plateau. Complementary to the controlled-rate measurements, stress-controlled measurementsshow that for a stress below the critical stress, the rate at which strain is acquired drops several orders of magnitude, providing evidence of a lower branch of the flow curve. In stress-controlled ageing experiments, the material recovers an increasing fraction of the strain acquired under stress with waiting time upon cessation of the (less than critical) stress. The freshly rejuvenated suspension recovers a mere 2%of the acquired strain, while for a waiting time of 104 s the material recovers 97% of the acquired strain. The material thus appears to evolve from a nearly ideal fluid to a nearly ideal solid. We relate this bulk evolution to spatially and temporally resolved Rheo-NMR velocity profiles which clearly show an evolution to a strongly shear-banded state. The velocity of the suspension in the lower shear band is below the uncertainty of the experiment. The growth of the (assumed) zero-shear band is well described by a Gompertz relation. The effects of shear-rate, temperature and waiting time on the Gompertzparameters are investigated. Phenomenological understanding is provided through a scalar model that describes the stress-dependent free-energy landscape. Using a dual-minimum free-energy landscape, the model is able to replicate the behaviour of the stress after startup in shearratecontrolled experiments, the flow curve and the velocity profiles across the gap of a Couette geometry. The Large-Amplitude-Oscillatory-Shear (LAOS) response is reported along with discussions of current LAOS analysis techniques. The stress response to LAOS of the star suspensions is well described in a Cox-Merz manner by a modified Cross model. The modified Cross model highlights an asymmetry in the LAOS response. This constitutes the first ever report of asymmetric LAOS responses. The asymmetry is followed as a function of time using two complementary scalar variables. A speculativeinterpretation is given to account for the evolution of the asymmetry.</p>

The Ageing and Rejuvenation of Soft, Glassy Complex Fluids

<p>Suspensions of multiarm star polymers are studied as models for soft colloidal interactionsin colloidal glasses. Establishing a pre-shearing protocol which ensures a reproducible initial state (the "rejuvenation" of the system), we report here the stress evolution from startup for two different concentrations for a range of shear rates using conventional rheological techniques. We show the existence of critical shear rateswhich are functions of the concentration. When the suspensions are sheared at rates below the critical rates, the stress rises to a common value which is also a function of the concentration. The system thus evolves into a yield stress-like fluid. This behavior manifests itself as an evolution from a monotonic, slightly shear-thinning flow curve to a flow curve dominated by a stress plateau. Complementary to the controlled-rate measurements, stress-controlled measurementsshow that for a stress below the critical stress, the rate at which strain is acquired drops several orders of magnitude, providing evidence of a lower branch of the flow curve. In stress-controlled ageing experiments, the material recovers an increasing fraction of the strain acquired under stress with waiting time upon cessation of the (less than critical) stress. The freshly rejuvenated suspension recovers a mere 2%of the acquired strain, while for a waiting time of 104 s the material recovers 97% of the acquired strain. The material thus appears to evolve from a nearly ideal fluid to a nearly ideal solid. We relate this bulk evolution to spatially and temporally resolved Rheo-NMR velocity profiles which clearly show an evolution to a strongly shear-banded state. The velocity of the suspension in the lower shear band is below the uncertainty of the experiment. The growth of the (assumed) zero-shear band is well described by a Gompertz relation. The effects of shear-rate, temperature and waiting time on the Gompertzparameters are investigated. Phenomenological understanding is provided through a scalar model that describes the stress-dependent free-energy landscape. Using a dual-minimum free-energy landscape, the model is able to replicate the behaviour of the stress after startup in shearratecontrolled experiments, the flow curve and the velocity profiles across the gap of a Couette geometry. The Large-Amplitude-Oscillatory-Shear (LAOS) response is reported along with discussions of current LAOS analysis techniques. The stress response to LAOS of the star suspensions is well described in a Cox-Merz manner by a modified Cross model. The modified Cross model highlights an asymmetry in the LAOS response. This constitutes the first ever report of asymmetric LAOS responses. The asymmetry is followed as a function of time using two complementary scalar variables. A speculativeinterpretation is given to account for the evolution of the asymmetry.</p>

The Influences of Sulphation, Salt Type, and Salt Concentration on the Structural Heterogeneity of Glycosaminoglycans

The increasing recognition of the biochemical importance of glycosaminoglycans (GAGs) has in recent times made them the center of attention of recent research investigations. It became evident that subtle conformational factors play an important role in determining the relationship between the chemical composition of GAGs and their activity. Therefore, a thorough understanding of their structural flexibility is needed, which is addressed in this work by means of all-atom molecular dynamics (MD) simulations. Four major GAGs with different substitution patterns, namely hyaluronic acid as unsulphated GAG, heparan-6-sulphate, chondroitin-4-sulphate, and chondroitin-6-sulphate, were investigated to elucidate the influence of sulphation on the dynamical features of GAGs. Moreover, the effects of increasing NaCl and KCl concentrations were studied as well. Different structural parameters were determined from the MD simulations, in combination with a presentation of the free energy landscape of the GAG conformations, which allowed us to unravel the conformational fingerprints unique to each GAG. The largest effects on the GAG structures were found for sulphation at position 6, as well as binding of the metal ions in the absence of chloride ions to the carboxylate and sulphate groups, which both increase the GAG conformational flexibility.