Functional stability of water wire-carbonyl interactions in an ion channel

Water wires are critical for the functioning of many membrane proteins, as in channels that conduct water, protons, and other ions. Here, in liquid crystalline lipid bilayers under symmetric environmental conditions, the selective hydrogen bonding interactions between eight waters comprising a water wire and a subset of 26 carbonyl oxygens lining the antiparallel dimeric gramicidin A channel are characterized by ¹⁷O NMR spectroscopy at 35.2 T (or 1,500 MHz for ¹H) and computational studies. While backbone ¹⁵N spectra clearly indicate structural symmetry between the two subunits, single site ¹⁷O labels of the pore-lining carbonyls report two resonances, implying a break in dimer symmetry caused by the selective interactions with the water wire. The ¹⁷O shifts document selective water hydrogen bonding with carbonyl oxygens that are stable on the millisecond timescale. Such interactions are supported by density functional theory calculations on snapshots taken from molecular dynamics simulations. Water hydrogen bonding in the pore is restricted to just three simultaneous interactions, unlike bulk water environs. The stability of the water wire orientation and its electric dipole leads to opposite charge-dipole interactions for K⁺ ions bound at the two ends of the pore, thereby providing a simple explanation for an ∼20-fold difference in K⁺ affinity between two binding sites that are ∼24 Å apart. The ¹⁷O NMR spectroscopy reported here represents a breakthrough in high field NMR technology that will have applications throughout molecular biophysics, because of the acute sensitivity of the ¹⁷O nucleus to its chemical environment.