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Author: search.filters.author.Prajapati, Jigneshkumar DahyabhaiSubject: search.filters.subject.Molecular dynamics simulations

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    Cyclodextrin interaction with specific channel CymA from K. Oxytoca
    (Cell Press, 2015-01) Prajapati, Jigneshkumar Dahyabhai
    The outer membrane acts as a selective uptake barrier in Gram negative bacteria. It contains protein channels (porins) which provide an entry pathway for hydrophilic molecules like small nutrient molecules and β-lactam antibiotics. However the CymA channel is known to take up cyclodextrin molecules giving bacteria the ability to survive on cyclodextrins. Hence understanding uptake of these molecules via porins is vital to comprehend the transport mechanism across the cell membrane. Electrophysiology forms a promising approach to study the permeation of molecules across outer membrane and thereby understanding molecular interactions with the channel. Here we present cyclodextrin interaction studies of CymA from K. oxytoca using single channel electrophysiology. Detailed single channel analysis revealed inherent asymmetric gating characteristics of the channel. Analysis of the ion current reduction through CymA in presence of cyclodextrin led revealed kinetic parameters of substrate binding. To further elucidate the affinity sites of substrate to the channel, mutation of certain channel residues has been performed. An altered channel gating behaviour is observed. To obtain an atomistic view we complement our studies with all-atom molecular dynamics simulation to study the various conductance states of the channel in the absence of cyclodextrin and to get molecular insight into the uptake of cyclodextrins as well.
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    Role of electroosmosis in the permeation of neutral molecules: CymA and cyclodextrin as an example
    (Cell Press, 2016-02) Prajapati, Jigneshkumar Dahyabhai
    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. 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.
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    Electro-osmotic driven kinetics of cyclodextrin through the cyma channel
    (Cell Press, 2016-02) Prajapati, Jigneshkumar Dahyabhai
    Trans membrane voltage is applied to understand the permeation of uncharged molecules into and across nano pores in Electrophysiology. The permeating molecule blocks the ion flow causing ion current fluctuations. However the fluctuation density is dependent on the magnitude and direction of the applied voltage. This dependency may be assumed due to electro-osmotic (EOF) flow, electrically driven ion – associated water flow. Here we investigate the contribution of the electro-osmotic flow (EOF) as a potential cause for an external voltage driven substrate permeation. As an example, we quantified the permeation of α-cyclodextrin through the cyclodextrin-specific channel-forming porin CymA from the Gram-negative bacterium Klebsiella oxytoca. To further elucidate these effects, substrate interaction studies were performed at various external voltages in presence of three different electrolyte solutions: KCl, NaCl and MgCl2. To demonstrate the significance of the EOF at an atomistic level, the net water flux was calculated and its effect on the binding affinity of substrates was studied by employing extensive molecular dynamics simulations.
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    Computational modeling of ion transport in bulk and through a nanopore using the drude polarizable force field
    (ACS, 2020-06) Prajapati, Jigneshkumar Dahyabhai
    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. However, the quality of these Drude FFs remains untested for such systems. Here, we examine the quality of this FF set in two ways, i.e., (i) in simple ionic aqueous solution simulations and (ii) in more complex membrane channel simulations. First, the aqueous solutions of KCl, NaCl, MgCl2, and CaCl2 salts are simulated using the polarizable Drude and the nonpolarizable CHARMM36 FFs. The bulk conductivity has been estimated for both FF sets using applied-field simulations for several concentrations and temperatures in the case of all investigated salts and compared to experimental findings. An excellent improvement in the ability of the Drude FF to reproduce the experimental bulk conductivities for KCl, NaCl, and MgCl2 solutions can be observed but not in the case of CaCl2. Moreover, the outer membrane channel OmpC from the bacterium Escherichia coli has been employed to examine the ability of the polarizable and nonpolarizable FFs to reproduce ion transport-related quantities known from experiment. Unbiased and applied-field simulations have been performed in the presence of KCl using both FF sets. Unlike for the bulk systems of aqueous salt solutions, it has been found that the Drude FF is not accurate in modeling KCl transport properties across the OmpC porin.