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Item Role of electroosmosis in the permeation of neutral molecules: CymA and cyclodextrin as an example(Cell Press, 2016-02) Prajapati, Jigneshkumar DahyabhaiTo 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. As a possible reason for this asymmetry, we identified the electroosmotic flow (EOF), which is the water transport associated with ions driven by the external transmembrane voltage. As an example, we quantify the contribution of the EOF through a nanopore by investigating the permeation of a-cyclodextrin through CymA, a cyclodextrin-specific channel from Klebsiella oxytoca. To understand the results from electrophysiology on a molecular level, all-atom molecular dynamics simulations are used to detail the effect of the EOF on substrate entry to and exit from a CymA channel in which the N-terminus has been deleted. The combined experimental and computational results strongly suggest that one needs to account for the significant contribution of the EOF when analyzing the penetration of cyclodextrins through the CymA pore. This example study at the same time points to the more general finding that the EOF needs to be considered in translocation studies of neutral molecules and, at least in many cases, should be able to help in discriminating between translocation and binding events.Item Effect of electroosmotic flow on the transport of α-cyclodextrin through the channel CymA(Elsevier, 2017-02) Prajapati, Jigneshkumar DahyabhaiCymA, an outer membrane channel of Klebsiella oxytoca, allows the passive diffusion of the bulky molecule α-cyclodextrin (α-CD, M.W. 972.8 Da) to the periplasm of the bacterium [1]. In single channel electrophysiology experiments, the flow of the uncharged α-CD was found asymmetric with respect to the applied voltage and ionic salts used. The net water current associated with the ion movement, i.e., the so-called electroosmotic flow (EOF), is induced by the ionic selectivity of the pore. This effects has been found to be a major factor behind the modified interactions of the α-CD molecule with the channel [2]. To get atomistic insight into the EOF on α-CD permeation, we have performed ∼40 μs free energy calculations in presence of three different ionic conditions, i.e., in the absence of ions, in the presence of 1 M KCl and of 1 M MgCl2, using well-tempered matadynamics simulations [3] applying an external field of 0 V, +1 V and −1 V. No major changes in the free energy landscapes were observed in the absence of ions at both polarities of the voltage. This finding indicates the absence of an electrophoretic effect on the neutral α-CD molecule and of an EOF mediated effect due to the absence ions. However, using an electric field together 1 M KCl salt, we observed significant free energy changes in the transport of the α-CD consistent with net EOF at both voltage polarities. Moreover, using 1 M MgCl2 salt, we demonstrate an alteration of the pore selectivity from cationic to anionic. Thereby, the direction of the resulting EOF at a particular voltage polarity and its effect on the α-CD permeation is inverted. These results highlight the role of the EOF in the transport of α-CD through the nanometer-sized CymA channel.