Probing the Transport of Ionic Liquids in Aqueous Solution through Nanopores
ABSTRACT 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.
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ABSTRACT: The TOM protein complex facilitates the transfer of nearly all mitochondrial preproteins across outer mitochondrial membranes. Here we characterized the effect of temperature on facilitated translocation of a mitochondrial presequence peptide pF1β. Ion current fluctuations analysis through single TOM channels revealed thermodynamic and kinetic parameters of substrate binding and allowed determining the energy profile of peptide translocation. The activation energy for the on-rate and off-rate of the presequence peptide into the TOM complex was symmetric with respect to the electric field and estimated to be about 15 and 22 kT per peptide. These values are above that expected for free diffusion of ions in water (6 kT) and reflect the stronger interaction in the channel. Both values are in the range for typical enzyme kinetics and suggest one process without involving large conformational changes within the channel protein. T he transfer of proteins across lipid membranes is a fundamental process in biology. Understanding the mechanism of this process has been one of the most challenging tasks of the past decade. Extensive studies of protein import into mitochondria in fungi, plants, and mammalian cells revealed several protein translocation machineries in inner and outer mitochondrial membranes. 1 In particular, the outer membrane preprotein translocase TOM acts as the main entry gate for nearly all proteins of mitochondria. Most components of the mitochondrial outer membrane import machinery are known and form multimeric protein complexes with a molecular mass between 350 and 500 kDa. 1−5 Electron microscopy studies on TOM purified from Neuro-spora crassa and yeast revealed the overall shape of the holo complex and the core complex, which consists of the components Tom40, 22, 7, 6, and 5. 2,5,6 Electrophysiological studies suggested that the general import pore Tom40 itself forms one pore. 6−8 Single-channel recordings of TOM core complex from Saccharomyces cerevisiae and N. crassa as well as isolated Tom40 reconstituted into planar lipid bilayers revealed a voltage-dependent increase of channel closures in the presence of mitochondrial targeting peptides. 7−12 However, since TOM channels show spontaneous voltage-dependent gating behavior in the absence of substrate, interactions between peptides and TOM were analyzed at low voltages only where endogenous channel gating was significantly reduced. 10,11 Here, we investigated the effect of temperature on peptide translocation through TOM core complex isolated and purified from N. crassa mitochondria (Supporting Information A). We selected a polypeptide corresponding to the first 31 residues of the mitochondrial precursor of the S. cerevisiae F1-ATPase β-subunit pF1β. 10,11 We observed temperature and voltage-dependent channel blockages in the presence of the mitochondrial presequence peptide pF1β at a single molecule level. An analysis of the temperature dependent rates gives information on the energy barrier for translocation. Isolated TOM core complex reconstituted in stable DPhPC lipid bilayers showed characteristic single channel conductance of ∼3.0 nS in 1 M KCl, 20 mM phosphate and pH 7.4 at room temperature. Increasing temperature cause the bulk conductance to increase. As expected, increasing the temperature from 2 to 30 °C increases the open channel conductance. Moreover, TOM channels show voltage and temperature-dependent channel closure. The higher the voltage and temperature, the more gating occurs (data not shown). TOM core complex showed an asymmetric channel closure and conductance with respect to the polarity of the applied voltage as shown previously. 10,11 At 2 °C (Figure 1A), the channel is open without any gating perturbations with stable ion current. Addition of peptide pF1β to the trans side produces few ion current blockages with typical blocking times of 1 ms at 2 °C (Figure 1A). Increasing the temperature to 10 °C results in an increase of the number of blockage events and a decrease of the residence time of peptide inside the channel (Figure 1B). At higher temperaturesJournal of Physical Chemistry Letters 12/2012; 4(1):78-82. · 6.59 Impact Factor
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ABSTRACT: Single channel electrophysiological studies have been carried out to elucidate the underlying interactions during the translocation of polypeptides through protein channels. For this we used OmpF from the outer cell membrane of E. coli and arginine-based peptides of different charges, lengths and covalently linked polyethylene glycol as a model system. In order to reveal the fast kinetics of peptide binding, we performed a temperature scan. Together with the voltage-dependent single-channel conductance, we quantify peptide binding and translocation.Biophysics of Structure and Mechanism 12/2012; · 2.44 Impact Factor