Peptide backbone chemistry and membrane channel function: Effects of a single amide-to-ester replacement on gramicidin channel structure and function

TO examine the structural and functional importance of backbone amide group s in ion channels for subunit folding, hydrogen bonding, ion solvation, and ion permeation, we replaced the peptide bond between Val(1) and Gly(2) in gramicidin A by an ester bond. The substitution is at the junction between the two channel subunits, where it removes an intramolecular hydrogen bond between the NH of Gly2 and the C=O of Val(7) and perturbs an intermolecular hydrogen bond between the C=O of Val(1) in one subunit and the NH of Ala(5 ) in the other subunit. The substitution thus perturbs not only subunit fol ding but also dimer assembly, in addition to any effects on ion permeation. This backbone modification has large effects on channel function: It alter s channel stability, as monitored by the channel forming ability and channe l lifetime, and ion permeability, as monitored by changes in single-channel conductance and cation permeability ratios. In fact, the homodimeric chann els, with two ester-containing subunits, have lifetimes so short that it be comes impossible to characterize them in any detail. The peptide -> ester s ubstitution, however, does not affect the basic subunit fold because hetero dimeric channels can form between a subunit with an eater bond and a native subunit. These heterodimeric channels, with only a single ester bond, are more easily characterized; the lone ester reduces the single-channel conduc tance about 4-fold and the lifetime about 200-fold as compared to the nativ e homodimeric channels. The altered channel function results from a perturb ation/disruption of the hydrogen bond network that stabilizes the backbone, as well as the membrane-spanning dimer, and that forms the lining of the i on-conducting pore. Molecular dynamics simulations show the expected destab ilization of the modified heterodimeric or homodimeric channels, but the ch anges in backbone structure and dynamics are remarkably small. The ester bo nd is somewhat unstable, which precluded further structural characterizatio n. The lability also led to a hydrolysis product that terminates with an al cohol and lacks formyl-Val. Symmetric channels formed by the hydrolyzed pro duct again have short lifetimes, but the channels are distinctly different from those formed by the ester gramicidin A. Furthermore, well-behaved asym metric channels form between the hydrolysis product and reference subunits that have either an L- or a D-residue at the formyl-NH-terminus.