The goal of this project is to evaluate computational methods for obtaining in-depth mechanistic insight into ion permeation by tightly combining information from experiments (electrophysiology, thermodynamics and NMR) and computations based on a hierarchical implementation of atomistic models (all-atom molecular dynamics, MD; multi-ion potential of mean force, PMF; and Brownian dynamics, BD). For such investigations to fully exploit the synergism between theory and experiment, the focus must be on a particularly well-defined ion channel. We selected the gramicidin channel because gramicidin channels are small enough that it is feasible to undertake detailed simulations, yet large enough that they have well-defined structural and functional properties - and they are non-trivial in the sense that their permeability properties have proven difficult to simulate quantitatively. Gramicidin channels thus become the system of choice for exploring whether it is possible to develop a comprehensive understanding of ion permeation at the molecular level using atomistic calculations. The proposed studies represent a cooperative effort among three laboratories, which provide complementary expertise to the project and have established records of productive collaborations. The initial computational studies will use a large existing base of measurements of single-channel currents as functions of permeant ion concentration and transmembrane voltage, as a reference for atomistic simulations of channel-mediated ion movement. In parallel, gramicidin analogues will be synthesized to introduce defined perturbations in the PMF for ion movement through the channels: Trp-""""""""Phe substitutions, where the polar Trp is replaced by the nonpolar Phe; Ala-""""""""Ser substitutions, where the non-polar Ala is replaced by the polar Ser. The structural consequences of the substitutions will be determined using solid-state NMR. The functional consequences will be determined using single-channel measurements. These analogues will be subject to MD and BD simulations - of the channel/side chain structure and dynamics, where the NMR results will serve as reference; and of ion permeation, where the electrophysiological results will serve as reference. The transfer free energy of alkali metal cations from water to N-methyl-acetamide will be measured and used to ascertain appropriate representation of ion-peptide group interactions and help calibrate/improve the atomic force field. The proposed research will help establish the range of validity of current models of ion permeation, identify the physical basis of any discrepancy, and implement solutions to resolve any difficulties that are encountered.
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