Structure-function correlations of an ion channel probed by solid state NMR

Ion channel selectivity and conductance efficiency are essential for biological function. High conductivity implies that the energy barriers along the transport pathway are fairly small and high selectivity depends on the differential difference in solvation energies in bulk solvent and in the channel for permeant and less permeant ions. It has been recognized that these two characteristics are in conflict with each other. In this work, orientational constraints including {dollar}sp{lcub}15{rcub}{dollar}N and {dollar}sp{lcub}13{rcub}{dollar}C chemical shifts, {dollar}sp2{dollar}H quadrupolar interactions, {dollar}sp{lcub}13{rcub}{dollar}C-{dollar}sp{lcub}15{rcub}{dollar}N and {dollar}sp{lcub}15{rcub}{dollar}N-{dollar}sp2{dollar}H dipolar interactions have been revisited by solid state NMR from uniformly aligned gramicidin A in hydrated lipid bilayers in the presence and absence of cations. The identified ion-channel interactions provide unique functional insight for how cationic selectivity is achieved without sacrifcing efficient conductance. The binding of cations to the gramicidin A channel appears to cause no significant structural deformation of either side chains or backbone. The monovalent cation binding site is indeed delocalized, which reduces the entropy penalty associated with cation binding. Dehydration occurs via an incremental process of replacing waters with carbonyl groups, and by so doing the association and dissociation rates for cation binding are high; an essential factor for efficient conductance. Flexibility in the monovalent cation solvation environment due to the water molecules accounts for the weak size selectivity of this channel and divalent cations are primarily excluded from entering this channel because of their high dehydration energy. Many of these principles are directly applicable to the recent. 3.2 A crystal structure of the K{dollar}sp+{dollar} channel where the resolution is not yet adequate. to make such detailed characterizations.; Transient dipolar oscillations in cross-polarized peptide in hydrated lipid samples provide a simple method for selectively polarizing spins and increasing the spectral sensitivity. Orientational constraints have been obtained from {dollar}sp{lcub}13{rcub}{dollar}C NMR with the suppression of the lipid carbonyl signals through selective polarization and spin exchange techniques. The observed {dollar}sp{lcub}13{rcub}{dollar}C{dollar}sb{lcub}alpha{rcub}{dollar}-{dollar}sp{lcub}13{rcub}{dollar}C{dollar}sb1{dollar} homonuclear dipolar splitting provides an alternative method for building up the three-dimensional protein structure and the very efficient carbon-carbon spin diffusion can potentially be used for resonance assignment. The two-dimensional Polarization Inversion Spin Exchange at Magic Angle (PISEMA) experiments have been used to derive higher resolution orientational constraints from unoriented hydrated samples.