Kinetic model for NS1643 drug activation of WT and L529I variants of Kv11.1 (hERG1) potassium channel.

Congenital and acquired (drug-induced) forms of the human long-QT syndrome are associated with alterations in Kv11.1 (hERG) channel-controlled repolarizing IKr currents of cardiac action potentials. A mandatory drug screen implemented by many countries led to a discovery of a large group of small molecules that can activate hERG currents and thus may act as potent antiarrhythmic agents. Despite significant progress in identification of channel activators, little is known about their mechanism of action. A combination of electrophysiological studies with molecular and kinetic modeling was used to examine the mechanism of a model activator (NS1643) action on the hERG channel and its L529I mutant. The L529I mutant has gating dynamics similar to that of wild-type while its response to application of NS1643 is markedly different. We propose a mechanism compatible with experiments in which the model activator binds to the closed (C3) and open states (O). We suggest that NS1643 is affecting early gating transitions, probably during movements of the voltage sensor that precede the opening of the activation gate.

BROMOC suite: Monte Carlo/Brownian dynamics suite for studies of ion permeation and DNA transport in biological and artificial pores with effective potentials.

The transport of ions and solutes by biological pores is central for cellular processes and has a variety of applications in modern biotechnology. The time scale involved in the polymer transport across a nanopore is beyond the accessibility of conventional MD simulations. Moreover, experimental studies lack sufficient resolution to provide details on the molecular underpinning of the transport mechanisms. BROMOC, the code presented herein, performs Brownian dynamics simulations, both serial and parallel, up to several milliseconds long. BROMOC can be used to model large biological systems. IMC-MACRO software allows for the development of effective potentials for solute-ion interactions based on radial distribution function from all-atom MD. BROMOC Suite also provides a versatile set of tools to do a wide variety of preprocessing and postsimulation analysis. We illustrate a potential application with ion and ssDNA transport in MspA nanopore.

Microsecond simulations of DNA and ion transport in nanopores with novel ion-ion and ion-nucleotides effective potentials.

We developed a novel scheme based on the grand-canonical Monte Carlo/Brownian dynamics simulations and have extended it to studies of ion currents across three nanopores with the potential for single-stranded DNA (ssDNA) sequencing: solid-state nanopore Si3N4, α-hemolysin, and E111N/M113Y/K147N mutant. To describe nucleotide-specific ion dynamics compatible with ssDNA coarse-grained model, we used the inverse Monte Carlo protocol, which maps the relevant ion-nucleotide distribution functions from all-atom molecular dynamics (MD) simulations. Combined with the previously developed simulation platform for Brownian dynamics simulations of ion transport, it allows for microsecond- and millisecond-long simulations of ssDNA dynamics in the nanopore with a conductance computation accuracy that equals or exceeds that of all-atom MD simulations. In spite of the simplifications, the protocol produces results that agree with the results of previous studies on ion conductance across open channels and provide direct correlations with experimentally measured blockade currents and ion conductances that have been estimated from all-atom MD simulations.

Dry excess electrons in room-temperature ionic liquids.

In this article, we describe general trends to be expected at short times when an excess electron is generated or injected in different room-temperature ionic liquids (RTILs). Perhaps surprisingly, the excess electron does not localize systematically on the positively charged cations. Rather, the excess charge localization pattern is determined by the cation and anion HOMO/LUMO gaps and, more importantly, by their relative LUMO alignments. As revealed by experiments, the short-time (ps/ns) transient UV spectrum of excess electrons in RTILs is often characterized by two bands, a broad band at low energies (above 1000 nm) and another weaker band at higher energies (around 400 nm). Our calculations show that the dry or presolvated electron spectrum (fs) also has two similar features. The broad band at low energies is due to transitions between electronic states with similar character on ions of the same class but in different locations of the liquid. The lower-intensity band at higher energies is due to transitions in which the electron is promoted to electronic states of different character, in some cases on counterions. Depending on the chemical nature of the RTIL, and especially on the anions, excess electrons can localize on cations or anions. Our findings hint at possible design strategies for controlling electron localization, where electron transfer or transport across species can be facilitated or blocked depending on the alignment of the electronic levels of the individual species.

Exploring the molecular basis of human manganese superoxide dismutase inactivation mediated by tyrosine 34 nitration.

Manganese Superoxide Dismutase (MnSOD) is an essential mitochondrial antioxidant enzyme that protects organisms against oxidative damage, dismutating superoxide radical (O2(.)⁻) into H2O2 and O2. The active site of the protein presents a Mn ion in a distorted trigonal-bipyramidal environment, coordinated by H26, H74, H163, D159 and one ⁻OH ion or H2O molecule. The catalytic cycle of the enzyme is a "ping-pong" mechanism involving Mn³+/Mn²+. It is known that nitration of Y34 is responsible for enzyme inactivation, and that this protein oxidative modification is found in tissues undergoing inflammatory and degenerative processes. However, the molecular basis about MnSOD tyrosine nitration affects the protein catalytic function is mostly unknown. In this work we strongly suggest, using computer simulation tools, that Y34 nitration affects protein function by restricting ligand access to the active site. In particular, deprotonation of 3-nitrotyrosine increases drastically the energetic barrier for ligand entry due to the absence of the proton. Our results for the WT and selected mutant proteins confirm that the phenolic moiety of Y34 plays a key role in assisting superoxide migration.

Molecular basis for the electric field modulation of cytochrome C structure and function.

Cytochrome c (Cyt) is a small soluble heme protein with a hexacoordinated heme and functions as an electron shuttle in the mitochondria and in early events of apoptosis when released to the cytoplasm. Using molecular dynamics simulations, we show here that biologically relevant electric fields induce an increased mobility and structural distortion of key protein segments that leads to the detachment of the sixth axial ligand Met80 from the heme iron. This electric-field-induced conformational transition is energetically and entropically driven and leads to a pentacoordinated high spin heme that is characterized by a drastically lowered reduction potential as well as by an increased peroxidase activity. The simulations provide a detailed atomistic picture of the structural effects of the electric field on the structure of Cyt, which allows a sound interpretation of recent experimental results. The observed conformational change may modulate the electron transfer reactions of Cyt in the mitochondria and, furthermore, may constitute a switch from the redox function in the respiratory chain to the peroxidase function in the early events of apoptosis.

Determinants of Isoform-Specific Gating Kinetics of hERG1 Channel: Combined Experimental and Simulation Study.

IKr is the rapidly activating component of the delayed rectifier potassium current, the ion current largely responsible for the repolarization of the cardiac action potential. Inherited forms of long QT syndrome (LQTS) (Lees-Miller et al., 1997) in humans are linked to functional modifications in the Kv11.1 (hERG) ion channel and potentially life threatening arrhythmias. There is little doubt now that hERG-related component of IKr in the heart depends on the tetrameric (homo- or hetero-) channels formed by two alternatively processed isoforms of hERG, termed hERG1a and hERG1b. Isoform composition (hERG1a- vs. the b-isoform) has recently been reported to alter pharmacologic responses to some hERG blockers and was proposed to be an essential factor pre-disposing patients for drug-induced QT prolongation. Very little is known about the gating and pharmacological properties of two isoforms in heart membranes. For example, how gating mechanisms of the hERG1a channels differ from that of hERG1b is still unknown. The mechanisms by which hERG 1a/1b hetero-tetramers contribute to function in the heart, or what role hERG1b might play in disease are all questions to be answered. Structurally, the two isoforms differ only in the N-terminal region located in the cytoplasm: hERG1b is 340 residues shorter than hERG1a and the initial 36 residues of hERG1b are unique to this isoform. In this study, we combined electrophysiological measurements for HEK cells, kinetics and structural modeling to tease out the individual contributions of each isoform to Action Potential formation and then make predictions about the effects of having various mixture ratios of the two isoforms. By coupling electrophysiological data with computational kinetic modeling, two proposed mechanisms of hERG gating in two homo-tetramers were examined. Sets of data from various experimental stimulation protocols (HEK cells) were analyzed simultaneously and fitted to Markov-chain models (M-models). The minimization procedure presented here, allowed assessment of suitability of different Markov model topologies and the corresponding parameters that describe the channel kinetics. The kinetics modeling pointed to key differences in the gating kinetics that were linked to the full channel structure. Interactions between soluble domains and the transmembrane part of the channel appeared to be critical determinants of the gating kinetics. The structures of the full channel in the open and closed states were compared for the first time using the recent Cryo-EM resolved structure for full open hERG channel and an homology model for the closed state, based on the highly homolog EAG1 channel. Key potential interactions which emphasize the importance of electrostatic interactions between N-PAS cap, S4-S5, and C-linker are suggested based on the structural analysis. The derived kinetic parameters were later used in higher order models of cells and tissue to track down the effect of varying the ratios of hERG1a and hERG1b on cardiac action potentials and computed electrocardiograms. Simulations suggest that the recovery from inactivation of hERG1b may contribute to its physiologic role of this isoform in the action potential. Finally, the results presented here contribute to the growing body of evidence that hERG1b significantly affects the generation of the cardiac Ikr and plays an important role in cardiac electrophysiology. We highlight the importance of carefully revisiting the Markov models previously proposed in order to properly account for the relative abundance of the hERG1 a- and b- isoforms.

BROMOCEA Code: An Improved Grand Canonical Monte Carlo/Brownian Dynamics Algorithm Including Explicit Atoms.

All-atom molecular dynamics simulations have a long history of applications studying ion and substrate permeation across biological and artificial pores. While offering unprecedented insights into the underpinning transport processes, MD simulations are limited in time-scales and ability to simulate physiological membrane potentials or asymmetric salt solutions and require substantial computational power. While several approaches to circumvent all of these limitations were developed, Brownian dynamics simulations remain an attractive option to the field. The main limitation, however, is an apparent lack of protein flexibility important for the accurate description of permeation events. In the present contribution, we report an extension of the Brownian dynamics scheme which includes conformational dynamics. To achieve this goal, the dynamics of amino-acid residues was incorporated into the many-body potential of mean force and into the Langevin equations of motion. The developed software solution, called BROMOCEA, was applied to ion transport through OmpC as a test case. Compared to fully atomistic simulations, the results show a clear improvement in the ratio of permeating anions and cations. The present tests strongly indicate that pore flexibility can enhance permeation properties which will become even more important in future applications to substrate translocation.

What controls open-pore and residual currents in the first sensing zone of alpha-hemolysin nanopore? Combined experimental and theoretical study.

The electrophoretic transport of single-stranded DNA through biological nanopores such as alpha-hemolysin (αHL) is a promising and cost-effective technology with the potential to revolutionize genomics. The rational design of pores with the controlled polymer translocation rates and high contrast between different nucleotides could improve significantly nanopore sequencing applications. Here, we apply a combination of theoretical and experimental methods in an attempt to elucidate several selective modifications in the pore which were proposed to be central for the effective discrimination between purines and pyrimidines. Our nanopore test set includes the wild type αHL and six mutants (E111N/M113X/K147N) in which the cross-section and chemical functionality of the first constriction zone of the pore are modified. Electrophysiological recordings were combined with all-atom Molecular Dynamics simulations (MD) and a recently developed Brownian Dynamics (BROMOC) protocol to investigate residual ion currents and pore-DNA interactions for two homo-polymers e.g. poly(dA)40 or poly(dC)40 blocking the pore. The calculated residual currents and contrast in the poly(dA)40/poly(dC)40 blocked pore are in qualitative agreement with the experimental recordings. We showed that a simple structural metric allows rationalization of key elements in the emergent contrast between purines and pyrimidines in the modified αHL mutants. The shape of the pore and its capacity for hydrogen bonding to a translocated polynucleotide are two essential parameters for contrast optimization. To further probe the impact of these two factors in the ssDNA sensing, we eliminated the effect of the primary constriction using serine substitutions (i.e. E111S/M113S/T145S/K147S) and increased the hydrophobic volume of the central residue in the secondary constriction (L135I). This pore modification sharply increased the contrast between Adenine (A) and Cytosine (C).

Effect of confinement on DNA, solvent and counterion dynamics in a model biological nanopore.

The application of recent advances in nanopore technology to high-throughput DNA sequencing requires a more detailed understanding of solvent, ion and DNA interactions occurring within these pores. Here we present a combination of atomistic and coarse-grained modeling studies of the dynamics of short single-stranded DNA (ssDNA) homopolymers within the alpha-hemolysin pore, for the two single-stranded homopolymers poly(dA)40 and poly(dC)40. Analysis of atomistic simulations along with the per-residue decomposition of protein-DNA interactions in these simulations gives new insight into the very complex issues that have yet to be fully addressed with detailed MD simulations. We discuss a modification of the solvent properties and ion distribution around DNA within nanopore confinement and put it into the general framework of counterion condensation theory. There is a reasonable agreement in computed properties from our all-atom simulations and the resulting predictions from analytical theories with experimental data, and our equilibrium results here support the conclusions from our previous non-equilibrium Brownian dynamics studies with a recently developed BROMOC protocol that cations are the primary charge carriers through alpha-hemolysin nanopores under an applied voltage in the presence of ssDNA. Clustering analysis led to an identification of distinct conformational states of captured polymer and depth of the current blockade. Therefore, our data suggest that confined polymer may act as a flickering gate, thus contributing to excess noise phenomena. We also discuss the extent of water structuring due to nanopore confinement and the relationship between the conformational dynamics of a captured polymer and the distribution of blocked current.

BROMOC-D: Brownian Dynamics/Monte-Carlo Program Suite to Study Ion and DNA Permeation in Nanopores.

A theoretical framework is presented to model ion and DNA translocation across a nanopore confinement under an applied electric field. A combined Grand Canonical Monte Carlo Brownian Dynamics (GCMC/BD) algorithm offers a general approach to study ion permeation through wide molecular pores with a direct account of ion-ion and ion-DNA correlations. This work extends previously developed theory by incorporating the recently developed coarse-grain polymer model of DNA by de Pablo and colleagues [Knotts, T. A.; Rathore, N.; Schwartz, D. C.; de Pablo, J. J. J. Chem. Phys. 2007, 126] with explicit ions for simulations of polymer dynamics. Atomistic MD simulations were used to guide model developments. The power of the developed scheme is illustrated with studies of single-stranded DNA (ss-DNA) oligomer translocation in two model cases: a cylindrical pore with a varying radius and a well-studied experimental system, the staphylococcal α-hemolysin channel. The developed model shows good agreement with experimental data for model studies of two homopolymers: ss-poly(dA)(n) and ss-poly(dC)(n). The developed protocol allows for direct evaluation of different factors (charge distribution and pore shape and size) controlling DNA translocation in a variety of nanopores.

What is the origin of the prepeak in the X-ray scattering of imidazolium-based room-temperature ionic liquids?

The observation of a first sharp diffraction peak (FSDP) at low frequency in the X-ray and neutron scattering spectra of different imidazolium-based room-temperature ionic liquids (RTILs) (the so-called prepeak) has often been experimentally interpreted as indicative of mesoscopic organization leading to nanoscale segregation and the formation of domains of different morphologies. This interpretation that has permeated the analysis of many recently published articles deserves an in depth theoretical analysis. In this article, we use several different computational techniques to thoroughly dissect the atomistic components giving rise to the low-frequency FSDP as well as other features in the structure function (S(q)). By understanding how S(q) changes as imidazolium-based ionic systems undergo solid-liquid phase transition, and by artificially perturbing the liquid structure in a way that directly couples to the intensity of the FSDP, we are able to identify in a rigorous way its geometric origin. Similar to the solid phase, the liquid phase is characterized by two typical length scales between polar groups. The shorter length scale gives rise to a shoulder peak in S(q) at about 0.9 Å(-1) whereas the longer one gives rise to the prepeak.

Modeling heme proteins using atomistic simulations.

Heme proteins are found in all living organisms, and perform a wide variety of tasks ranging from electron transport, to the oxidation of organic compounds, to the sensing and transport of small molecules. In this work we review the application of classical and quantum-mechanical atomistic simulation tools to the investigation of several relevant issues in heme proteins chemistry: (i) conformational analysis, ligand migration, and solvation effects studied using classical molecular dynamics simulations; (ii) electronic structure and spin state energetics of the active sites explored using quantum-mechanics (QM) methods; (iii) the interaction of heme proteins with small ligands studied through hybrid quantum mechanics-molecular mechanics (QM-MM) techniques; (iv) and finally chemical reactivity and catalysis tackled by a combination of quantum and classical tools.

Mechanisms of NO release by N1-nitrosomelatonin: nucleophilic attack versus reducing pathways.

A new type of physiologically relevant nitrosamines have been recently recognized, the N(1)-nitrosoindoles. The possible pathways by which N(1)-nitrosomelatonin (NOMel) can react in physiological environments have been studied. Our results show that NOMel slowly decomposes spontaneously in aqueous solution, generating melatonin as the main organic product (k = (3.7 +/- 1.1) x 10(-5) s(-1), Tris-HCl (0.2 M) buffer, pH 7.4 at 37 degrees C, anaerobic). This rate is accelerated by acidification (k(pH 5.8) = (4.5 +/- 0.7) x 10(-4) s(-1), k(pH 8.8) = (3.9 +/- 0.6) x 10(-6) s(-1), Tris-HCl (0.2 M) buffer at 37 degrees C), by the presence of O(2) (k(o) = (9.8 +/- 0.1) x 10(-5) s(-1), pH 7.4, 37 degrees C, [NOMel] = 0.1 mM, P(O(2)) = 1 atm), and by the presence of the spin trap TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl; k(o) = (2.0 +/- 0.1) x 10(-4) s(-1), pH 7.4, 37 degrees C, [NOMel] = 0.1 mM, [TEMPO] = 9 mM). We also found that NOMel can transnitrosate to l-cysteinate, producing S-nitrosocysteine and melatonin (k = 0.127 +/- 0.002 M(-1) s(-1), Tris-HCl (0.2 M) buffer, pH 7.4 at 37 degrees C). The reaction of NOMel with ascorbic acid as a reducing agent has also been studied. This rapid reaction produces nitric oxide and melatonin. The saturation of the observed rate constant (k = (1.08 +/- 0.04) x 10(-3) s(-1), Tris-HCl (0.2 M) buffer, pH 7.4 at 37 degrees C) at high ascorbic acid concentration (100-fold with respect to NOMel) and the pH independence of this reaction in the pH range 7-9 indicate that the reactive species are ascorbate and melatonyl radical originated from the reversible homolysis of NOMel. Taking into account kinetic and DFT calculation data, a comprehensive mechanism for the denitrosation of NOMel is proposed. On the basis of our kinetics results, we conclude that under physiological conditions NOMel mainly reacts with endogenous reducing agents (such as ascorbic acid), producing nitric oxide and melatonin.