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Item Cyclodextrin interaction with specific channel CymA from K. Oxytoca(Cell Press, 2015-01) Prajapati, Jigneshkumar DahyabhaiThe 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.Item Atomistic modeling of two-dimensional electronic spectra and excited-state dynamics for a light harvesting 2 complex(ACS, 2015-01) Prajapati, Jigneshkumar DahyabhaiThe Light Harvesting 2 (LH2) complex is a vital part of the photosystem of purple bacteria. It is responsible for the absorption of light and transport of the resulting excitations to the reaction center in a highly efficient manner. A general description of the chromophores and the interaction with their local environment is crucial to understand this highly efficient energy transport. Here we include this interaction in an atomistic way using mixed quantum-classical (molecular dynamics) simulations of spectra. In particular, we present the first atomistic simulation of nonlinear optical spectra for LH2 and use it to study the energy transport within the complex. We show that the frequency distributions of the pigments strongly depend on their positions with respect to the protein scaffold and dynamics of their local environment. Furthermore, we show that although the pigments are closely packed the transition frequencies of neighboring pigments are essentially uncorrelated. We present the simulated linear absorption spectra for the LH2 complex and provide a detailed explanation of the states responsible for the observed two-band structure. Finally, we discuss the energy transfer within the complex by analyzing population transfer calculations and 2D spectra for different waiting times. We conclude that the energy transfer from the B800 ring to the B850 ring is mediated by intermediate states that are delocalized over both rings, allowing for a stepwise downhill energy transport.Item Outer-membrane translocation of bulky small molecules by passive diffusion(Proceedings of the National Academy of Sciences, 2015-04) Prajapati, Jigneshkumar DahyabhaiThe 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. Intriguingly, the OM protein CymA from Klebsiella oxytoca does not depend on TonB but nevertheless mediates efficient OM passage of cyclodextrins with diameters of up to ∼15 Å. Here we show, by using X-ray crystallography, molecular dynamics simulations, and single-channel electrophysiology, that CymA forms a monomeric 14-stranded β-barrel with a large pore that is occluded on the periplasmic side by the N-terminal 15 residues of the protein. Representing a previously unidentified paradigm in OM transport, CymA mediates the passive diffusion of bulky molecules via an elegant transport mechanism in which a mobile element formed by the N terminus acts as a ligand-expelled gate to preserve the permeability barrier of the OM.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 Electro-osmotic driven kinetics of cyclodextrin through the cyma channel(Cell Press, 2016-02) Prajapati, Jigneshkumar DahyabhaiTrans 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.Item Biophysical insight into the substrate permeation through the major outer membrane channels of acinetobacter baumannii(Cell Press, 2017-02) Prajapati, Jigneshkumar DahyabhaiAmong other mechanisms the cause for Multidrug Resistant (MDR) bacteria is their reduced permeability for antibiotics in particular in Gram-negative bacteria such as Acinetobacter baumanii. Owing to the low permeability (100 fold less compared to that of E. coli) and genetic plasticity to adopt to the external environment accompanied with robust efflux pumps aids these bugs in limiting the intracellular active concentration of the antibiotic to minimum. Here we characterize transport of small water soluble molecules across channels in the outer membrane (OM) of A. baumanii. We use Single Channel Electrophysiology as a main tool for the biophysical characterization of the majorly expressed channels from A. baumanii. Combining our study with high resolution X- ray crystallography and molecular dynamics simulations, we provide insight into the OM of A. baumanii.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.Item Characterization of ciprofloxacin permeation pathways across the Porin OmpC using metadynamics and a string method(ACS, 2017-08) Prajapati, Jigneshkumar DahyabhaiThe 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. Here, a theoretical study concerning the diffusion route of ciprofloxacin across the outer membrane porin OmpC from E. coli is presented. To this end, we establish a protocol to characterize meaningful permeation pathways by combining metadynamics with the zero-temperature string method. It was found that the lowest-energy pathway requires a reorientation of ciprofloxacin in the extracellular side of the porin before reaching the constriction region with its carboxyl group ahead. Several affinity sites have been identified, and their metastability has been evaluated using unbiased simulations. Such a detailed understanding is potentially very helpful in guiding the development of next generation antibiotics.Item Enrofloxacin permeation pathways across the porin OmpC(ACS, 2018-01) Prajapati, Jigneshkumar DahyabhaiIn 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. D. Prajapati et al. J. Chem. Theory Comput. 2017, 13, 4553], we characterized the diffusion pathway of a ciprofloxacin molecule through the outer membrane porin OmpC of Escherichia coli by combining metadynamics and a zero-temperature string method. Here, we evaluate the diffusion route through the OmpC porin for a similar fluoroquinolone, that is, the enrofloxacin molecule, using the previously developed protocol. As a result, it was found that the lowest-energy pathway was similar to that for ciprofloxacin; namely, a reorientation was required on the extracellular side with the carboxyl group ahead before enrofloxacin reached the constriction region. In turn, the free-energy basins for both antibiotics are located at similar positions in the space defined by selected reaction coordinates, and their affinity sites share a wide number of porin residues. However, there are some important deviations due to the chemical differences of these two drugs. On the one hand, a slower diffusion process is expected for enrofloxacin, as the permeation pathway exhibits higher overall energy barriers, mainly in the constriction region. On the other hand, enrofloxacin needs to replace some polar interactions in its affinity sites with nonpolar ones. This study demonstrates how minor chemical modifications can qualitatively affect the translocation mechanism of an antibiotic molecule.Item Environmental effects on the dynamics in the light-harvesting complexes LH2 and LH3 based on molecular simulations(Elsevier, 2018-11) Prajapati, Jigneshkumar DahyabhaiAlthough a multitude of theoretical studies exist on light-harvesting complex 2 (LH2), less is known about the light-harvesting complex 3 (LH3) of similar ring-like structure. In a comparative study of three system, i.e., the LH2 protein-pigment aggregate of the purple bacterium Rhodospirillum molischianum as well as for the LH2 and LH3 complexes of Rhodoblastus acidophilus the similarities and the differences in the excitonic system were analyzed. To this end, the systems have been studied in a multi-scale approach that combines molecular dynamics simulations with quantum chemistry calculations and quantum dynamics. Along the ground-state molecular dynamics trajectories, the excited energy gaps were determined in a quantum mechanics/molecular mechanics hybrid fashion. Based on the simulations, spectral densities, absorption spectra and exciton dynamics have been determined. After correcting for some shortcoming in the absorption spectra, the exciton dynamics within and between the ring systems have been determined and discussed.