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Purpose: Early detection of neurofibromatosis type 1 (NF1)-associated peripheral nerve sheath tumors (PNST) informs clinical decision-making, enabling early definitive treatment and potentially averting deadly outcomes. In this study, we describe a cell-free DNA (cfDNA) fragmentomic approach that distinguishes nonmalignant, premalignant, and malignant forms of PNST in the cancer predisposition syndrome, NF1., Experimental Design: cfDNA was isolated from plasma samples of a novel cohort of 101 patients with NF1 and 21 healthy controls and underwent whole-genome sequencing. We investigated diagnosis-specific signatures of copy-number alterations with in silico size selection as well as fragment profiles. Fragmentomics were analyzed using complementary feature types: bin-wise fragment size ratios, end motifs, and fragment non-negative matrix factorization signatures., Results: The novel cohort of patients with NF1 validated that our previous cfDNA copy-number alteration-based approach identifies malignant PNST (MPNST) but cannot distinguish between benign and premalignant states. Fragmentomic methods were able to differentiate premalignant states including atypical neurofibromas (AN). Fragmentomics also adjudicated AN cases suspicious for MPNST, correctly diagnosing samples noninvasively, which could have informed clinical management., Conclusions: Novel cfDNA fragmentomic signatures distinguish AN from benign plexiform neurofibromas and MPNST, enabling more precise clinical diagnosis and management. This study pioneers the early detection of malignant and premalignant PNST in NF1 and provides a blueprint for decentralizing noninvasive cancer surveillance in hereditary cancer predisposition syndromes., (©2024 The Authors; Published by the American Association for Cancer Research.)

To perform their physiological functions, amino methyl propionic acid receptors (AMPARs) cycle through active, resting, and desensitized states, and dysfunction in AMPAR activity is associated with various neurological disorders. Transitions among AMPAR functional states, however, are largely uncharacterized at atomic resolution and are difficult to examine experimentally. Here, we report long-timescale molecular dynamics simulations of dimerized AMPAR ligand-binding domains (LBDs), whose conformational changes are tightly coupled to changes in AMPAR functional states, in which we observed LBD dimer activation and deactivation upon ligand binding and unbinding at atomic resolution. Importantly, we observed the ligand-bound LBD dimer transition from the active conformation to several other conformations, which may correspond with distinct desensitized conformations. We also identified a linker region whose structural rearrangements heavily affected the transitions to and among these putative desensitized conformations, and confirmed, using electrophysiology experiments, the importance of the linker region in these functional transitions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Intrinsically disordered proteins (IDPs) are implicated in many human diseases. They have generally not been amenable to conventional structure-based drug design, however, because their intrinsic conformational variability has precluded an atomic-level understanding of their binding to small molecules. Here we present long-time-scale, atomic-level molecular dynamics (MD) simulations of monomeric α-synuclein (an IDP whose aggregation is associated with Parkinson's disease) binding the small-molecule drug fasudil in which the observed protein-ligand interactions were found to be in good agreement with previously reported NMR chemical shift data. In our simulations, fasudil, when bound, favored certain charge-charge and π-stacking interactions near the C terminus of α-synuclein but tended not to form these interactions simultaneously, rather breaking one of these interactions and forming another nearby (a mechanism we term dynamic shuttling ). Further simulations with small molecules chosen to modify these interactions yielded binding affinities and key structural features of binding consistent with subsequent NMR experiments, suggesting the potential for MD-based strategies to facilitate the rational design of small molecules that bind with disordered proteins.

Management of infections in patients with cystic fibrosis (CF) presents challenges for healthcare providers, including the eradication of initial acquisition, treatment of acute exacerbations, and chronic infection with suppressive therapy. Inhaled antimicrobial therapy for infections in patients with CF has been used in these capacities, often in an effort to achieve optimal concentrations in sputum for antimicrobial efficacy while mitigating potential toxicities associated with systemic therapy. Unfortunately, there are few commercially available products formulated for inhalation, resulting in the off-label use of other formulations, such as intravenous products, administered via nebulization. This review aims to examine the evidence supporting the efficacy of these off-label formulations for management of acute and chronic infections associated with CF, as well as adverse effects associated with their use., (© 2019 Wiley Periodicals, Inc.)

Bruton's tyrosine kinase (Btk) is critical for B cell proliferation and activation, and the development of Btk inhibitors is a vigorously pursued strategy for the treatment of various B cell malignancies. A detailed mechanistic understanding of Btk activation has, however, been lacking. Here, inspired by a previous suggestion that Btk activation might depend on dimerization of its lipid-binding PH-TH module on the cell membrane, we performed long-timescale molecular dynamics simulations of membrane-bound PH-TH modules and observed that they dimerized into a single predominant conformation. We found that the phospholipid PIP 3 stabilized the dimer allosterically by binding at multiple sites, and that the effects of PH-TH mutations on dimer stability were consistent with their known effects on Btk activity. Taken together, our simulation results strongly suggest that PIP 3 -mediated dimerization of Btk at the cell membrane is a critical step in Btk activation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Despite the biological importance of protein-protein complexes, determining their structures and association mechanisms remains an outstanding challenge. Here, we report the results of atomic-level simulations in which we observed five protein-protein pairs repeatedly associate to, and dissociate from, their experimentally determined native complexes using a molecular dynamics (MD)-based sampling approach that does not make use of any prior structural information about the complexes. To study association mechanisms, we performed additional, conventional MD simulations, in which we observed numerous spontaneous association events. A shared feature of native association for these five structurally and functionally diverse protein systems was that if the proteins made contact far from the native interface, the native state was reached by dissociation and eventual reassociation near the native interface, rather than by extensive interfacial exploration while the proteins remained in contact. At the transition state (the conformational ensemble from which association to the native complex and dissociation are equally likely), the protein-protein interfaces were still highly hydrated, and no more than 20% of native contacts had formed., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The pathways that G protein-coupled receptor (GPCR) ligands follow as they bind to or dissociate from their receptors are largely unknown. Protease-activated receptor-1 (PAR1) is a GPCR activated by intramolecular binding of a tethered agonist peptide that is exposed by thrombin cleavage. By contrast, the PAR1 antagonist vorapaxar is a lipophilic drug that binds in a pocket almost entirely occluded from the extracellular solvent. The binding and dissociation pathway of vorapaxar is unknown. Starting with the crystal structure of vorapaxar bound to PAR1, we performed temperature-accelerated molecular dynamics simulations of ligand dissociation. In the majority of simulations, vorapaxar exited the receptor laterally into the lipid bilayer through openings in the transmembrane helix (TM) bundle. Prior to full dissociation, vorapaxar paused in metastable intermediates stabilized by interactions with the receptor and lipid headgroups. Derivatives of vorapaxar with alkyl chains predicted to extend between TM6 and TM7 into the lipid bilayer inhibited PAR1 with apparent on rates similar to that of the parent compound in cell signaling assays. These data are consistent with vorapaxar binding to PAR1 via a pathway that passes between TM6 and TM7 from the lipid bilayer, in agreement with the most consistent pathway observed by molecular dynamics. While there is some evidence of entry of the ligand into rhodopsin and lipid-activated GPCRs from the cell membrane, our study provides the first such evidence for a peptide-activated GPCR and suggests that metastable intermediates along drug binding and dissociation pathways can be stabilized by specific interactions between lipids and the ligand.

Fibrils and oligomers are the aggregated protein agents of neuronal dysfunction in ALS diseases. Whereas we now know much about fibril architecture, atomic structures of disease-related oligomers have eluded determination. Here, we determine the corkscrew-like structure of a cytotoxic segment of superoxide dismutase 1 (SOD1) in its oligomeric state. Mutations that prevent formation of this structure eliminate cytotoxicity of the segment in isolation as well as cytotoxicity of the ALS-linked mutants of SOD1 in primary motor neurons and in a Danio rerio (zebrafish) model of ALS. Cytotoxicity assays suggest that toxicity is a property of soluble oligomers, and not large insoluble aggregates. Our work adds to evidence that the toxic oligomeric entities in protein aggregation diseases contain antiparallel, out-of-register β-sheet structures and identifies a target for structure-based therapeutics in ALS., Competing Interests: The authors declare no conflict of interest.

A quantitative characterization of the binding properties of drug fragments to a target protein is an important component of a fragment-based drug discovery program. Fragments typically have a weak binding affinity, however, making it challenging to experimentally characterize key binding properties, including binding sites, poses, and affinities. Direct simulation of the binding equilibrium by molecular dynamics (MD) simulations can provide a computational route to characterize fragment binding, but this approach is so computationally intensive that it has thus far remained relatively unexplored. Here, we perform MD simulations of sufficient length to observe several different fragments spontaneously and repeatedly bind to and unbind from the protein FKBP, allowing the binding affinities, on- and off-rates, and relative occupancies of alternative binding sites and alternative poses within each binding site to be estimated, thereby illustrating the potential of long time scale MD as a quantitative tool for fragment-based drug discovery. The data from the long time scale fragment binding simulations reported here also provide a useful benchmark for testing alternative computational methods aimed at characterizing fragment binding properties. As an example, we calculated binding affinities for the same fragments using a standard free energy perturbation approach and found that the values agreed with those obtained from the fragment binding simulations within statistical error.

How drugs dissociate from their targets is largely unknown. We investigated the molecular basis of this process in the adenosine A2Areceptor (A2AR), a prototypical G protein-coupled receptor (GPCR). Through kinetic radioligand binding experiments, we characterized mutant receptors selected based on molecular dynamic simulations of the antagonist ZM241385 dissociating from the A2AR. We discovered mutations that dramatically altered the ligand's dissociation rate despite only marginally influencing its binding affinity, demonstrating that even receptor features with little contribution to affinity may prove critical to the dissociation process. Our results also suggest that ZM241385 follows a multistep dissociation pathway, consecutively interacting with distinct receptor regions, a mechanism that may also be common to many other GPCRs., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Molecular dynamics (MD) simulations can describe protein motions in atomic detail, but transitions between protein conformational states sometimes take place on time scales that are infeasible or very expensive to reach by direct simulation. Enhanced sampling methods, the aim of which is to increase the sampling efficiency of MD simulations, have thus been extensively employed. The effectiveness of such methods when applied to complex biological systems like proteins, however, has been difficult to establish because even enhanced sampling simulations of such systems do not typically reach time scales at which convergence is extensive enough to reliably quantify sampling efficiency. Here, we obtain sufficiently converged simulations of three proteins to evaluate the performance of simulated tempering, a member of a widely used class of enhanced sampling methods that use elevated temperature to accelerate sampling. Simulated tempering simulations with individual lengths of up to 100 μs were compared to (previously published) conventional MD simulations with individual lengths of up to 1 ms. With two proteins, BPTI and ubiquitin, we evaluated the efficiency of sampling of conformational states near the native state, and for the third, the villin headpiece, we examined the rate of folding and unfolding. Our comparisons demonstrate that simulated tempering can consistently achieve a substantial sampling speedup of an order of magnitude or more relative to conventional MD.

Structurally elucidating transition pathways between protein conformations gives deep mechanistic insight into protein behavior but is typically difficult. Unbiased molecular dynamics (MD) simulations provide one solution, but their computational expense is often prohibitive, motivating the development of enhanced sampling methods that accelerate conformational changes in a given direction, embodied in a collective variable. The accuracy of such methods is unclear for complex protein transitions, because obtaining unbiased MD data for comparison is difficult. Here, we use long-time scale, unbiased MD simulations of epidermal growth factor receptor kinase deactivation as a complex biological test case for two widely used methods-steered molecular dynamics (SMD) and the string method. We found that common collective variable choices, based on the root-mean-square deviation (RMSD) of the entire protein, prevented the methods from producing accurate paths, even in SMD simulations on the time scale of the unbiased transition. Using collective variables based on the RMSD of the region of the protein known to be important for the conformational change, however, enabled both methods to provide a more accurate description of the pathway in a fraction of the simulation time required to observe the unbiased transition.

The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, 'orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

Understanding the nature of the glass transition--the dramatic slowing of dynamics and eventual emergence of a disordered solid from a cooling liquid--is a fundamental challenge in physical science. A central characteristic of glass-forming liquids is a non-exponential main relaxation process. The extent of deviation from exponential relaxation typically becomes more pronounced on cooling. Theories that predict a growth of spatially heterogeneous dynamics as temperature is lowered can explain these observations. In apparent contradiction to these theories, however, some experiments suggest that certain substances--notably including the intensely studied molecular glass-former ortho-terphenyl (OTP)--have a main relaxation process whose shape is essentially temperature independent, even though other observables predicted to be correlated with the degree of dynamical heterogeneity are temperature dependent. Here we report the first simulations based on an atomistic model of OTP that reach equilibrium at temperatures well into the supercooled regime. We first show that the results of these simulations are in reasonable quantitative agreement with experimental data for several basic properties over a wide range of temperatures. We then focus on rotational relaxation, finding nearly exponential behavior at high temperatures with clearly increasing deviations as temperature is lowered. The much weaker temperature dependence observed in light-scattering experiments also emerges from the same simulation data when we calculate correlation functions similar to those probed experimentally; this highlights the diversity of temperature dependencies that can be obtained with different probes. Further analysis suggests that the temperature insensitivity observed in the light-scattering experiments stems from the dependence of these measurements on internal as well as rotational molecular motion. Within the temperature range of our OTP simulations, our results strongly suggest that this archetypal glass-former behaves as anticipated by theories of the glass transition that predict increasing non-exponentiality with cooling, and our simulations thus strengthen the evidence supporting such theories.

Application of a specific stimulus opens the intracellular gate of a K(+) channel (activation), yielding a transient period of ion conduction until the selectivity filter spontaneously undergoes a conformational change towards a non-conductive state (inactivation). Removal of the stimulus closes the gate and allows the selectivity filter to interconvert back to its conductive conformation (recovery). Given that the structural differences between the conductive and inactivated filter are very small, it is unclear why the recovery process can take up to several seconds. The bacterial K(+) channel KcsA from Streptomyces lividans can be used to help elucidate questions about channel inactivation and recovery at the atomic level. Although KcsA contains only a pore domain, without voltage-sensing machinery, it has the structural elements necessary for ion conduction, activation and inactivation. Here we reveal, by means of a series of long molecular dynamics simulations, how the selectivity filter is sterically locked in the inactive conformation by buried water molecules bound behind the selectivity filter. Potential of mean force calculations show how the recovery process is affected by the buried water molecules and the rebinding of an external K(+) ion. A kinetic model deduced from the simulations shows how releasing the buried water molecules can stretch the timescale of recovery to seconds. This leads to the prediction that reducing the occupancy of the buried water molecules by imposing a high osmotic stress should accelerate the rate of recovery, which was verified experimentally by measuring the recovery rate in the presence of a 2-molar sucrose concentration.

It is increasingly appreciated that the rates at which drugs associate with and dissociate from receptors--the binding kinetics--directly impact drug efficacy and safety. The molecular determinants of drug-receptor binding kinetics remain poorly understood, however, especially when compared with the well-known factors that affect binding affinity. The rational modulation of kinetics during lead optimization thus remains challenging. We review some of the key factors thought to control drug-receptor binding kinetics at the molecular level--molecular size, conformational fluctuations, electrostatic interactions and hydrophobic effects--and discuss several possible approaches for the rational design of drugs with desired binding kinetics., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The epidermal growth factor receptor (EGFR) is a key protein in cellular signaling, and its kinase domain (EGFR kinase) is an intensely pursued target of small-molecule drugs. Although both catalytically active and inactive conformations of EGFR kinase have been resolved crystallographically, experimental characterization of the transitions between these conformations remains difficult. Using unbiased, all-atom molecular dynamics simulations, we observed EGFR kinase spontaneously transition from the active to the so-called "Src-like inactive" conformation by way of two sets of intermediate conformations: One corresponds to a previously identified locally disordered state and the other to previously undescribed "extended" conformations, marked by the opening of the ATP-binding site between the two lobes of the kinase domain. We also simulated the protonation-dependent transition of EGFR kinase to another ["Asp-Phe-Gly-out" ("DFG-out")] inactive conformation and observed similar intermediate conformations. A key element observed in the simulated transitions is local unfolding, or "cracking," which supports a prediction of energy landscape theory. We used hydrogen-deuterium (H/D) exchange measurements to corroborate our simulations and found that the simulated intermediate conformations correlate better with the H/D exchange data than existing active or inactive EGFR kinase crystal structures. The intermediate conformations revealed by our simulations of the transition process differ significantly from the existing crystal structures and may provide unique possibilities for structure-based drug discovery.

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β(2)-adrenergic receptor (β(2)AR), a prototypical GPCR. We labeled β(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for β(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for β(2)AR's ability to engage multiple signaling and regulatory proteins., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In K+ channels, rearrangements of the pore outer vestibule have been associated with C-type inactivation gating. Paradoxically, the crystal structure of Open/C-type inactivated KcsA suggests these movements to be modest in magnitude. In this study, we show that under physiological conditions, the KcsA outer vestibule undergoes relatively large dynamic rearrangements upon inactivation. External Cd2+ enhances the rate of C-type inactivation in an cysteine mutant (Y82C) via metal-bridge formation. This effect is not present in a non-inactivating mutant (E71A/Y82C). Tandem dimer and tandem tetramer constructs of equivalent cysteine mutants in KcsA and Shaker K+ channels demonstrate that these Cd2+ metal bridges are formed only between adjacent subunits. This is well supported by molecular dynamics simulations. Based on the crystal structure of Cd2+ -bound Y82C-KcsA in the closed state, together with electron paramagnetic resonance distance measurements in the KcsA outer vestibule, we suggest that subunits must dynamically come in close proximity as the channels undergo inactivation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G(q/11)-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G(i/o)-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

The amount of ionic current flowing through K(+) channels is determined by the interplay between two separate time-dependent processes: activation and inactivation gating. Activation is concerned with the stimulus-dependent opening of the main intracellular gate, whereas inactivation is a spontaneous conformational transition of the selectivity filter toward a nonconductive state occurring on a variety of timescales. A recent analysis of multiple x-ray structures of open and partially open KcsA channels revealed the mechanism by which movements of the inner activation gate, formed by the inner helices from the four subunits of the pore domain, bias the conformational changes at the selectivity filter toward a nonconductive inactivated state. This analysis highlighted the important role of Phe103, a residue located along the inner helix, near the hinge position associated with the opening of the intracellular gate. In the present study, we use free energy perturbation molecular dynamics simulations (FEP/MD) to quantitatively elucidate the thermodynamic basis for the coupling between the intracellular gate and the selectivity filter. The results of the FEP/MD calculations are in good agreement with experiments, and further analysis of the repulsive, van der Waals dispersive, and electrostatic free energy contributions reveals that the energetic basis underlying the absence of inactivation in the F103A mutation in KcsA is the absence of the unfavorable steric interaction occurring with the large Ile100 side chain in a neighboring subunit when the intracellular gate is open and the selectivity filter is in a conductive conformation. Macroscopic current analysis shows that the I100A mutant indeed relieves inactivation in KcsA, but to a lesser extent than the F103A mutant.

A third of marketed drugs act by binding to a G-protein-coupled receptor (GPCR) and either triggering or preventing receptor activation. Although recent crystal structures have provided snapshots of both active and inactive functional states of GPCRs, these structures do not reveal the mechanism by which GPCRs transition between these states. Here we propose an activation mechanism for the β(2)-adrenergic receptor, a prototypical GPCR, based on atomic-level simulations in which an agonist-bound receptor transitions spontaneously from the active to the inactive crystallographically observed conformation. A loosely coupled allosteric network, comprising three regions that can each switch individually between multiple distinct conformations, links small perturbations at the extracellular drug-binding site to large conformational changes at the intracellular G-protein-binding site. Our simulations also exhibit an intermediate that may represent a receptor conformation to which a G protein binds during activation, and suggest that the first structural changes during receptor activation often take place on the intracellular side of the receptor, far from the drug-binding site. By capturing this fundamental signaling process in atomic detail, our results may provide a foundation for the design of drugs that control receptor signaling more precisely by stabilizing specific receptor conformations.

How drugs bind to their receptors--from initial association, through drug entry into the binding pocket, to adoption of the final bound conformation, or "pose"--has remained unknown, even for G-protein-coupled receptor modulators, which constitute one-third of all marketed drugs. We captured this pharmaceutically critical process in atomic detail using the first unbiased molecular dynamics simulations in which drug molecules spontaneously associate with G-protein-coupled receptors to achieve final poses matching those determined crystallographically. We found that several beta blockers and a beta agonist all traverse the same well-defined, dominant pathway as they bind to the β(1)- and β(2)-adrenergic receptors, initially making contact with a vestibule on each receptor's extracellular surface. Surprisingly, association with this vestibule, at a distance of 15 Å from the binding pocket, often presents the largest energetic barrier to binding, despite the fact that subsequent entry into the binding pocket requires the receptor to deform and the drug to squeeze through a narrow passage. The early barrier appears to reflect the substantial dehydration that takes place as the drug associates with the vestibule. Our atomic-level description of the binding process suggests opportunities for allosteric modulation and provides a structural foundation for future optimization of drug-receptor binding and unbinding rates.

Modal-gating shifts represent an effective regulatory mechanism by which ion channels control the extent and time course of ionic fluxes. Under steady-state conditions, the K(+) channel KcsA shows three distinct gating modes, high-P(o), low-P(o) and a high-frequency flicker mode, each with about an order of magnitude difference in their mean open times. Here we show that in the absence of C-type inactivation, mutations at the pore-helix position Glu71 unmask a series of kinetically distinct modes of gating in a side chain-specific way. These gating modes mirror those seen in wild-type channels and suggest that specific interactions in the side chain network surrounding the selectivity filter, in concert with ion occupancy, alter the relative stability of pre-existing conformational states of the pore. The present results highlight the key role of the selectivity filter in regulating modal gating behavior in K(+) channels.

The coupled interplay between activation and inactivation gating is a functional hallmark of K(+) channels. This coupling has been experimentally demonstrated through ion interaction effects and cysteine accessibility, and is associated with a well defined boundary of energetically coupled residues. The structure of the K(+) channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation-inactivation gating. Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter, allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K(+) channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K(+) channels.

An efficient method is proposed for building Markov models with discrete states able to accurately describe the slow relaxation of a complex system with two stable conformations. First, the reaction pathway described by a set of collective variables between the two stable states is determined using the string method with swarms of trajectories. Then, short trajectories are initiated at different points along this pathway to build the state-to-state transition probability matrix. It is shown, using a model system, how this strategy makes it possible to use trajectories that are significantly shorter than the slowest relaxation time to efficiently build a reliable and accurate Markov model. Extensions of the method to multiple pathways, as well as some common pitfalls arising from poorly relaxed paths or an inappropriate choice of collective variables, are illustrated and discussed.

An approach to find transition pathways in complex systems is presented. The method, which is related to the string method in collective variables of Maragliano et al. (J. Chem. Phys. 2006, 125, 024106), is conceptually simple and straightforward to implement. It consists of refining a putative transition path in the multidimensional space supported by a set of collective variables using the average dynamic drift of those variables. This drift is estimated on-the-fly via swarms of short unbiased trajectories started at different points along the path. Successive iterations of this algorithm, which can be naturally distributed over many computer nodes with negligible interprocessor communication, refine an initial trial path toward the most probable transition path (MPTP) between two stable basins. The method is first tested by determining the pathway for the C7eq to C7ax transition in an all-atom model of the alanine dipeptide in vacuum, which has been studied previously with the string method in collective variables. A transition path is found with a committor distribution peaked at 1/2 near the free energy maximum, in accord with previous results. Last, the method is applied to the allosteric conformational change in the nitrogen regulatory protein C (NtrC), represented here with a two-state elastic network model. Even though more than 550 collective variables are used to describe the conformational change, the path converges rapidly. Again, the committor distribution is found to be peaked around 1/2 near the free energy maximum between the two stable states, confirming that a genuine transition state has been localized in this complex multidimensional system.

Dynamical four-point susceptibilities measure the extent of spatial correlations in the dynamics of glass forming systems. We show how these susceptibilities depend on the lengthscales that necessarily form part of their definition. The behavior of these susceptibilities is estimated by means of an analysis in terms of renewal processes within the context of dynamic facilitation. The analytic results are confirmed by numerical simulations of an atomistic model glass former, and of two kinetically constrained models. Hence we argue that the scenario predicted by the dynamic facilitation approach is generic.

We have used a combination of neutron scattering experiments and Monte Carlo simulations to study the initial stages of first-order phase transitions. We focus on quenches wherein the nascent phase is formed by homogeneous nucleation, and we approach the spinodal, i.e., the quench depth at which the original phase becomes unstable. In this regime, we show how critical nuclei sizes are determined from neutron scattering structure factors. Prevailing thought is that the size of the critical nucleus should increase with increasing quench depth and diverge at the spinodal. To the contrary, our experiments and simulations indicate that the critical nucleus size decreases monotonically as quench depth is increased and is finite at the spinodal.

We study the dynamical heterogeneity and glassy dynamics in a kinetically constrained lattice-gas model which has both translational and rotational degrees of freedom. We find that the rotational relaxation time tracks the structural relaxation time as density is increased whereas the translational diffusion constant exhibits a strong decoupling. We investigate distributions of exchange and persistence times for both the rotational and translational degrees of freedom and compare our results on the distributions of rotational exchange times to recent single molecule studies.

We study dynamical heterogeneity and growing dynamical length scales in two kinetically constrained models, namely, the one- and two-vacancy assisted triangular lattice gases. One of the models is a strong glassformer and the other is a fragile glassformer. Both exhibit heterogeneous dynamics with broadly distributed time scales as seen in the distribution of persistence times. We show that the Stokes-Einstein relation is violated, to a greater degree in the fragile glassformer, and show how this violation is related to dynamic heterogeneity. We extract dynamical length scales from structure factors of mobile particles and show, quantitatively, the growth of this length scale as density increases. We comment on how the scaling of lengths and times in these models relates to that in facilitated spin models of glasses.

Background: Conventional pathogenesis of haemorrhoid emphasized the anchoring connective tissue system, whereas the vascular changes were ignored. The aim of this study was to clarify vascular changes of haemorrhoid disease., Materials and Methods: Forty-six samples of grade III and grade IV haemorrhoid tissue were selected for an in vitro study. We assessed the expressions in endoglin (CD105), an accessory protein in transforming growth factor-beta receptor complex, in CD34 and in vascular endothelial growth factor by using an immunohistochemical method. Microvascular density was calculated to correlate the expression of endoglin., Results: Microvascular density was higher in haemorrhoid tissue than in normal anal and lower rectal tissues. CD34 was demonstrated in whole vessels in the haemorrhoids. However, endoglin, a proliferative marker of neovascularization, was present in only 25 of 46 (54%) haemorrhoidal vessels, and its immunoactivity was prominent in venules larger than 100 micro m. Thrombosis formation and stromal vascular endothelial growth factor was significantly associated with the presence of endoglin immunoactivity., Conclusion: The results of this study suggest that neovascularization is one important phenomenon of haemorrhoid disease, along with conventional venous dilatation and arteriovenous communication. In addition, thrombosis and stromal vascular endothelial growth factor might be important factors in promoting vascular proliferation.