Probing the Transport of Ionic Liquids in Aqueous Solution through Nanopores

The temperature-dependent transport of the ionic liquid 1-butyl-3-methyl-imidazolium chloride (BMIM-Cl) in aqueous solution is studied theoretically and experimentally. Using molecular dynamics simulations and ion-conductance measurements, the transport is examined in bulk as well as through a biological nanopore, that is, OmpF and its mutant D113A. This investigation is motivated by the observation that aqueous solutions of BMIM-Cl drastically reduce the translocation speed of DNA or antibiotics through nanopores in electrophysiological measurements. This makes BMIM-Cl an interesting alternative salt to improve the time resolution. In line with previous investigations of simple salts, the size of the ions and their orientation adds another important degree of freedom to the ion transport, thereby slowing the transport through nanopores. An excellent agreement between theory and conductance measurements is obtained for wild type OmpF and a reasonable agreement for the mutant. Moreover, all-atom simulations allow an atomistic analysis revealing molecular details of the rate-limiting ion interactions with the channel

Systematic Data Mining Reveals Synergistic H3R/MCHR1 Ligands

In this study, we report a ligand-centric data mining approach that guided the identification of suitable target profiles for treating obesity. The newly developed method is based on identifying target pairs for synergistic positive effects and also encompasses the exclusion of compounds showing a detrimental effect on obesity treatment (off-targets). Ligands with known activity against obesity-relevant targets were compared using fingerprint representations. Similar compounds with activities to different targets were evaluated for the mechanism of action since activation or deactivation of drug targets determines the pharmacological effect. <i>In vitro</i> validation of the modeling results revealed that three known modulators of melanin-concentrating hormone receptor 1 (MCHR1) show a previously unknown submicromolar affinity to the histamine H3 receptor (H₃R). This synergistic activity may present a novel therapeutic option against obesity

Analysis of PorACj purification.

<p>(A) Western blot analysis illustrating IMAC purification of his-tagged PorACj protein. The protein was expressed in <i>C. glutamicum</i> ATCC13032 <i>ΔporHΔporA</i> and purified by Ni²⁺ affinity from the supernatant of detergent extracted whole cells. CMDIE represents chloroform-methanol treated cells in which the crude protein content was concentrated around 8 fold by diethyl-ether precipitation of pXJK0268His transfected (+) or non-transfected (−) <i>C. glutamicumΔporHΔporA</i> cells. Subsequent to tricine (12%)-SDS-PAGE the gel was blotted on a nitrocellulose membrane and PorACj-His was visualized by Anti-His antibodies and a chemiluminescent reaction. All samples were boiled for 5 minutes in Redmix before loading. (B) Silver stained tricine (16.5%)-SDS-PAGE of Ni²⁺-purified and factor Xa digested PorACj-His protein. Lanes: 1, 3 units of protease Xa (control); 2, 10 µl of three pooled Ni-NTA elution containing PorACj-His; 3, 10 µl of protease Xa treated and purified PorACj protein (for details see text). The dot blot immunoassay pictures underneath lanes 2 and 3 show cleavage of the histidine tail using anti-his antibody of 5 µl of the corresponding protein samples. Before loading all samples were boiled for 5 minutes in Redmix.</p

Analysis of secondary structure of PorACj usinf CD-spectrometry.

<p>A: CD spectra of recombinant PorACj (69 µM) and PorA-His₈ (12 µM) solubilized in 0.5% Genapol, 100 mM NaCl, 50 mM TrisHCl and 1 mM CaCl₂, pH 8 measured at room temperature. B: CD-spectra of the same protein samples as in (A). The aqueous solutions of the proteins was supplemented with 4 M urea to destroy the secondary structure of the proteins.</p

Conductance (G) at a given membrane potential (V_m) divided by the conductance at 10

<p> <b>mV (G₀) expressed as a function of the membrane potential.</b> The symbols represent the mean (± SD) of six measurements, in which pure PorACj protein was added to the <i>cis</i>-side of the membranes. The aqueous phase contained 1 M KCl and 100 ng/ml porin. The membranes were formed from PC/<i>n</i>-decane at a temperature of 20°C.</p

Analysis of PorACj secondary structure.

<p>(A) The panel shows the hydrophobicity indices of the individual amino acids of PorACj according to ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075651#pone.0075651-Kyte1" target="_blank">[80]</a>. (B) The secondary structure of PorACj was predicted using a consensus method [83] at the Pole Bioinformatique Lyonnaise network (<a href="http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html" target="_blank">http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html</a>); the protein was suggested to form α-helices. Amino acid residues arranged on basis of heptameric repeats (a–g) showing distinct separation in a hydrophobic domain supposable surrounded by lipid molecules (dark grey) while the hydrophilic domain (light grey) is suggested to represent the component orientated to the water-filled lumen in the presumed oligomeric PorACj.</p

Dendrogram representing the phylogenetic relationships of PorA and PorH of different <i>Corynebacterium</i> species obtained by the neighbor-joining method.

<p>The tree was derived from the alignments of corresponding gene sequences. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node (in percent).The software used to construct alignment and tree was MEGA5.1.The sequence was aligned by ClustalW. Parameters: Multiple Alignment: Gap Opening Penalty: 10; Gap Extension Penalty: 0.2; Protein Weight Matrix: Gonnet; Gap Separation Distance: 4; Delay Divergent Cutoff (%): 30; The phylogenetic tree of corynebacterial species was constructed using the Maximum Likelihood statistical method; Substitution Model; Substitutions Type: Amino acid; Model/Method: Jones-Taylor-Thornton (JTT) model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075651#pone.0075651-Tamura1" target="_blank">[81]</a>.</p

Minimum inhibitory concentration (MIC) and diameters of inhibition zones of antimicrobial agents for <i>C. glutamicum</i> ΔHA pXjk0268His and <i>C. glutamicum</i> ΔHA as control.

<p>NI means no inhibition of growth, i.e. no growth inhibition zone.</p

Investigation of the voltage-dependence of PorACj in a multi-channel experiment.

<p>The purified protein was added to the <i>cis</i>-side of a PC membrane (100 ng/ml) and the reconstitution of channels was followed until equilibrium. Then increasing positive (upper traces) and negative voltages (lower traces) were applied to the <i>cis</i>-side of the membrane, and the membrane current was measured as a function of time. The aqueous phase contained 1 M KCl; T = 20°C.</p

Study of pore-forming capacity of purified PorACj.

<p>(A) Single-channel recording of a PC/<i>n</i>-decane membrane in the presence of pure PorACj. The aqueous phase contained 1 M KCl, pH 6 and 10 ng/ml protein. The applied membrane potential was 20 mV; T = 20^°C. (B) Histogram of the probability P(G) for the occurrence of a given conductivity unit observed with membranes formed of 1% PC dissolved in <i>n</i>-decane. It was calculated by dividing the number of fluctuations with a given conductance rise by the total number of conductance fluctuations in the presence of pure PorACj. Two frequent conductive units were observed for 295 single events taken from eight individual membranes. The average conductance of the steps corresponding to the left-side maximum was 1.25 nS and that of the right-side maximum was 2.5 nS. The aqueous phase contained 1 M KCl, pH 6 and 10 ng/ml protein, the applied membrane potential was 20 mV, T = 20°C.</p