Magnesium ions mitigate metastable states in the regulatory landscape of mRNA elements.

Residing in the 5' untranslated region of the mRNA, the 2'-deoxyguanosine (2'-dG) riboswitch mRNA element adopts an alternative structure upon binding of the 2'-dG molecule, which terminates transcription. RNA conformations are generally strongly affected by positively charged metal ions (especially Mg). We have quantitatively explored the combined effect of ligand (2'-dG) and Mg binding on the energy landscape of the aptamer domain of the 2'-dG riboswitch with both explicit solvent all-atom molecular dynamics simulations (99 μsec aggregate sampling for the study) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments.

Conformational Dynamics of Loop L3 in OmpF: Implications toward Antibiotic Translocation and Voltage Gating.

In the present work, we delineate the molecular mechanism of a bulky antibiotic permeating through a bacterial channel and uncover the role of conformational dynamics of the constriction loop in this process. Using the temperature accelerated sliced sampling approach, we shed light onto the dynamics of the L3 loop, in particular the F118 to S125 segment, at the constriction regions of the OmpF porin. We complement the findings with single channel electrophysiology experiments and applied-field simulations, and we demonstrate the role of hydrogen-bond stabilization in the conformational dynamics of the L3 loop.

Exploring the Energy Landscape of Riboswitches Using Collective Variables Based on Tertiary Contacts.

Messenger RNA regulatory elements, such as riboswitches, can display a high degree of flexibility. By characterizing their energy landscapes, and corresponding distributions of 3D configurations, structure-function relationships can be elucidated. Molecular dynamics simulation with enhanced sampling is an important strategy used to computationally access free energy landscapes characterizing the accessible 3D conformations of RNAs.

Identification of Single Amino Acid Chiral and Positional Isomers Using an Electrostatically Asymmetric Nanopore.

Chirality is essential in nearly all biological organizations and chemical reactions but is rarely considered due to technical limitations in identifying L/D isomerization. Using OmpF, a membrane channel from with an electrostatically asymmetric constriction zone, allows discriminating chiral amino acids in a single peptide. The heterogeneous distribution of charged residues in OmpF causes a strong lateral electrostatic field at the constriction.

Atomistic Simulation of Molecules Interacting with Biological Nanopores: From Current Understanding to Future Directions.

Biological nanopores have been at the focus of numerous studies due to their role in many biological processes as well as their (prospective) technological applications. Among many other topics, recent studies on nanopores have addressed two key areas: antibiotic permeation through bacterial channels and sensing of analytes. Although the two areas are quite far apart in terms of their objectives, in both cases atomistic simulations attempt to understand the solute dynamics and the solute-protein interactions within the channel lumen.

Changes in Salt Concentration Modify the Translocation of Neutral Molecules through a ΔCymA Nanopore in a Non-monotonic Manner.

The voltage-dependent transport through biological and artificial nanopores is being used in many applications such as DNA or protein sequencing and sensing. The primary approach to determine the transport has been to measure the temporal ion current fluctuations caused by solutes when applying external voltages. Crossing the nanoscale confinement in the presence of an applied electric field primarily relies on two factors, i.

Improved Sampling and Free Energy Estimates for Antibiotic Permeation through Bacterial Porins.

Antibiotics enter into bacterial cells via protein channels that serve as low-energy pathways through the outer membrane, which is otherwise impenetrable. Insights into the molecular mechanisms underlying the transport processes are vital for the development of effective antibacterials. A much-desired prerequisite is an accurate and reproducible determination of free energy surfaces for antibiotic translocation, enabling quantitative and meaningful comparisons of permeation mechanisms for different classes of antibiotics.

Dynamic interaction of fluoroquinolones with magnesium ions monitored using bacterial outer membrane nanopores.

Divalent ions are known to have a severe effect on the translocation of several antibiotic molecules into (pathogenic) bacteria. In the present study we have investigated the effect of divalent ions on the permeability of norfloxacin across the major outer membrane channels from (OmpF and OmpC) and (Omp35 and Omp36) at the single channel level. To understand the rate limiting steps in permeation, we reconstituted single porins into planar lipid bilayers and analyzed the ion current fluctuations caused in the presence of norfloxacin.

How to Enter a Bacterium: Bacterial Porins and the Permeation of Antibiotics.

Despite tremendous successes in the field of antibiotic discovery seen in the previous century, infectious diseases have remained a leading cause of death. More specifically, pathogenic Gram-negative bacteria have become a global threat due to their extraordinary ability to acquire resistance against any clinically available antibiotic, thus urging for the discovery of novel antibacterial agents. One major challenge is to design new antibiotics molecules able to rapidly penetrate Gram-negative bacteria in order to achieve a lethal intracellular drug accumulation.

Large-Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca*.

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter-pair-based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation through CymA is the rate-limiting factor.

Millisecond-Long Simulations of Antibiotics Transport through Outer Membrane Channels.

To reach their target site inside Gram-negative bacteria, almost all antibiotics need to cross the outer membrane. Computational modeling of such processes can be numerically demanding due to the size of the systems and especially due to the timescales involved. Recently, a hybrid Brownian and molecular dynamics approach, i.

Voltage-Dependent Transport of Neutral Solutes through Nanopores: A Molecular View.

The permeation of (neutral) molecules through nanopores in the presence of external voltages depends on several factors including pore electrostatics, electrophoretic force, and electro-osmotic drag. In earlier single-channel electrophysiology experiments, voltage-dependent asymmetric transport of neutral α-cyclodextrin (α-CD) molecules through the biological nanopore ΔCymA was observed. The voltage-dependent ion-associated flow of water, the so-called electro-osmotic flow, has been suggested to be the key factor behind the observed asymmetric behavior.

DFTB/MM Molecular Dynamics Simulations of the FMO Light-Harvesting Complex.

Because of the size of light-harvesting complexes and the involvement of electronic degrees of freedom, computationally these systems need to be treated with a combined quantum-classical description. To this end, Born-Oppenheimer molecular dynamics simulations have been employed in a quantum mechanics/molecular mechanics (QM/MM) fashion for the ground state followed by excitation energy calculations again in a QM/MM scheme for the Fenna-Matthews-Olson (FMO) complex. The self-consistent-charge density functional tight-binding (DFTB) method electrostatically coupled to a classical description of the environment was applied to perform the ground-state dynamics.

Computational Modeling of Ion Transport in Bulk and through a Nanopore Using the Drude Polarizable Force Field.

In the past two decades, molecular dynamics simulations have become the method of choice for elucidating the transport mechanisms of ions through various membrane channels. Often, these simulations heavily rely on classical nonpolarizable force fields (FFs), which lack electronic polarizability in the treatment of the electrostatics. The recent advancements in the Drude polarizable FF lead to a complete set of parameters for water, ions, protein, and lipids, allowing for a more realistic modeling of membrane proteins.

Exploration of Free Energy Surfaces Across a Membrane Channel Using Metadynamics and Umbrella Sampling.

To reach their site of action, it is essential for antibiotic molecules to cross the bacterial outer membrane. The progress of enhanced sampling techniques in molecular dynamics simulations enables us to understand these translocations at an atomic level. To this end, calculations of free energy surfaces for these permeation processes are of key importance.

Molecular Interactions of Cephalosporins with the Deep Binding Pocket of the RND Transporter AcrB.

The drug/proton antiporter AcrB, part of the major efflux pump AcrABZ-TolC in Escherichia coli, is characterized by its impressive ability to transport chemically diverse compounds, conferring a multidrug resistance phenotype. However, the molecular features differentiating between good and poor substrates of the pump have yet to be identified. In this work, we combined molecular docking with molecular dynamics simulations to study the interactions between AcrB and two representative cephalosporins, cefepime and ceftazidime (a good and poor substrate of AcrB, respectively).

A Multidisciplinary Approach toward Identification of Antibiotic Scaffolds for Acinetobacter baumannii.

Research efforts to discover potential new antibiotics for Gram-negative bacteria suffer from high attrition rates due to the synergistic action of efflux systems and the limited permeability of the outer membrane (OM). One strategy to overcome the OM permeability barrier is to identify small molecules that are natural substrates for abundant OM channels and use such compounds as scaffolds for the design of efficiently permeating antibacterials. Here we present a multidisciplinary approach to identify such potential small-molecule scaffolds.

Enrofloxacin Permeation Pathways across the Porin OmpC.

In Gram-negative bacteria, the lack or quenching of antibiotic translocation across the outer membrane is one of the main factors for acquiring antibiotic resistance. An atomic-level comprehension of the key features governing the transport of drugs by outer-membrane protein channels would be very helpful in developing the next generation of antibiotics. In a previous study [ J.

Characterization of Ciprofloxacin Permeation Pathways across the Porin OmpC Using Metadynamics and a String Method.

The rapid spreading of antimicrobial resistance in Gram-negative bacteria has become a major threat for humans as well as animals. As one of the main factors involved, the permeability of the outer membrane has attracted a great deal of attention recently. However, the knowledge regarding the translocation mechanisms for most available antibiotics is so far rather limited.

Role of Electroosmosis in the Permeation of Neutral Molecules: CymA and Cyclodextrin as an Example.

To quantify the flow of small uncharged molecules into and across nanopores, one often uses ion currents. The respective ion-current fluctuations caused by the presence of the analyte make it possible to draw some conclusions about the direction and magnitude of the analyte flow. However, often this flow appears to be asymmetric with respect to the applied voltage.

Outer-membrane translocation of bulky small molecules by passive diffusion.

The outer membrane (OM) of gram-negative bacteria forms a protective layer around the cell that serves as a permeability barrier to prevent unrestricted access of noxious substances. The permeability barrier of the OM results partly from the limited pore diameters of OM diffusion channels. As a consequence, there is an "OM size-exclusion limit," and the uptake of bulky molecules with molecular masses of more than ∼ 600 Da is thought to be mediated by TonB-dependent, active transporters.