Location of ion-binding sites in the gramicidin channel by X-ray diffraction

We report the first X-ray diffraction on gramicidin in its membrane-active form by using uniformly aligned multilayer samples of membranes containing gramicidin and ions (Tl +, K +, Ba 2+, Mg 2+ or without ions). Prom the difference electron density profiles, we found a pair of symmetrically located ion-binding sites for Tl + at 9.6(±0.3) Å and for Ba 2+ at 13.0(±0.2) Å from the midpoint of the gramicidin channel. The location of Ba 2+-binding sites is near the ends of the channel, consistent with the experimental observation that divalent cations do not permeate but block the channel. The location of Tl +-binding sites is somewhat of a surprise. It was generally thought that monovalent cations bind to the first turn of the helix from the mouth of the channel. (It is now generally accepted that the gramicidin channel is a cylindrical pore formed by two monomers, each a single-stranded β 6.3 helix and hydrogen-bonded head-to-head at their N termini.) But our experiment shows that the Tl +-binding site is either near the bottom of or below the first helix turn.