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Studying the migration of an excess proton or a fluoride anion is crucial to understanding mechanisms of many physiological processes, as they generally involve ion transport across a channel or a transporter. However, the molecular nature of ion transport is complicated, involving Grotthuss shuttling mechanism and/or electron polarization effect. In this dissertation, MS-RMD is employed to systematically investigate the proton solvation and transport mechanism through a Cl–/H+ antiporter ClC-ec1, a protein that stoichiometrically exchange Cl– and H+ ions in opposite directions across a membrane. It has been shown that alternative polyatomic anion flux including NO3– and SCN– or site-specific mutation (e.g., the I109F mutation) can partially or completely block the proton flux. With the help of multiscale computer simulations, it is demonstrated how the chemical nature of these anions alters the coupling mechanism and qualitatively explain the shifts in the ion stoichiometry. Multidimensional free energy profiles for PT and the coupled changes in hydration are presented for NO3– and SCN–. The calculated proton flux agrees with the experiments, showing reduced or abolished rate. We suggested that the size of anions and interactions with the protein significantly alter hydration and shift its influence on PT from facilitating to inhibiting, and we revealed that the most relevant coordinate to the PT free energy barrier is the water connectivity along the PT pathway which is significantly affected by the nature of the bound anion. In addition, reactive molecular dynamics simulations of explicit proton transport across the central region in the I109F mutant has also been performed, and a two-dimensional free energy profile has been constructed that is consistent with the experimental transport rates. The importance of a phenylalanine gate formed by F109 and F357 and its influence on hydration connectivity through the central proton transport pathway is revealed. Our work demonstrates how seemingly subtle changes in local conformational dynamics can dictate hydration changes and thus transport properties. In the following chapter of the dissertation, we present our work of employing the CHARMM-Drude model to investigate the fluoride transport mechanism through the Ec2 channel, which transports fluoride at the rate of 106–107 s−1. It has been shown that conserved residues N41, F80, F83, H106 as well as an interfacial Na+ are crucial to the F– transport. Our constant-pH molecular dynamics simulations reveal that F– is deprotonated while His106 remains protonated. Following replica-exchange umbrella sampling (REUS) simulations using both nonpolarizable and polarizable models indicates the resulting diffusion constant as well as rate constant from polarizable simulations matches the experimental measurements better, implying the necessity of a polarizable force field. Our simulations suggest several F– binding states, which reveal the electrostatic attraction between F– and Na+, Phe C–H···F hydrogen bonds, a H106A side-chain rotation, and a F83A–H106A gate, explaining aforementioned experimental discoveries. These binding states also provide insights on following mutagenetic experiments on conserved residues R19 and T37. These findings help to explain the F− transport mechanism in Ec2 as well as other Fluc proteins.



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