The Membrane Interface Dictates Different Anchor Roles for “Inner Pair” and “Outer Pair” Tryptophan Indole Rings in Gramicidin A Channels

Abstract

We investigated the effects of substituting two of the four tryptophans (the “inner pair” Trp9,11 or the “outer pair” Trp13,15) in gramicidin A (gA) channels. The conformational preferences of the double-substituted gA analogues were assessed using circular dichroism spectroscopy and size-exclusion chromatography, which show that the inner tryptophans 9 and 11 are critical for the gA’s conformational preference in lipid bilayer membranes. [Phe13,15]gA largely retains the single-stranded helical channel structure, whereas of [Phe9,11]gA exists primarily as double-stranded conformers. Within this context, the 2H-NMR spectra from labeled tryptophans were used to examine the changes in average indole ring orientations, induced by the Phe substitutions and by the shift in conformational preference. Using a method for deuterium labeling of already synthesized gAs, we introduced deuterium selectively onto positions C2 and C5 of the remaining tryptophan indole rings in the substituted gA analogues for solid-state 2H-NMR spectroscopy. The (least possible) changes in orientation and overall motion of each indole ring were estimated from the experimental spectra. Regardless of the mixture of backbone folds, the indole ring orientations observed in the analogues are similar to those found previously for gA channels. Both Phe-substituted analogues form single-stranded channels, as judged from the formation of heterodimeric channels with the native gA. [Phe13,15]gA channels have Na+ currents that are ~50% and lifetimes ~80% those of native gA channels. The double-stranded conformer(s) of [Phe9,11]gA do not form detectable channels. The minor single-stranded population of [Phe9,11]gA forms channels with Na+ currents that are ~25% and single-channel lifetimes that are ~300% those of native gA channels. Our results suggest that Trp9 and Trp11, when “reaching” for the interface, tend to drive both monomer folding (to “open” a channel) and dimer dissociation (to “close” a channel). Furthermore, the dipoles of Trp9 and Trp11 are relatively more important for the single-channel conductance than are the dipoles of Trp13 and Trp15.

Importance of indole NH hydrogen bonding in the organization and dynamics of gramicidin channels

“…In addition, we have recently shown that the positions of tryptophan residues in membranes are important for gramicidin structure and function [37,54]. We show here that the hydrogen bonding ability of tryptophans represents a crucial determinant in maintaining the gramicidin channel structure in the membrane.…”

“…Circular dichroism spectroscopy is a convenient method to monitor conformations of gramicidin in membranes [25,36,43,54]. CD spectrum for the channel conformation of gramicidin displays two characteristic peaks of positive ellipticity at ~218 and 235 nm, a valley at 230 nm, and negative ellipticity below 208 nm.…”

Conformation of gramicidin A in Triton X‐100 micelles from CD and FTIR data: a clean example of antiparallel double β5.6 helix formation

“…Tryptophan residues have been previously shown to be of crucial importance for manifestation of the channel function of gA . As mentioned in the Results section, the CD intensity in the B b band (228 nm) of Trp indole in the CD spectrum of the ↑↓β5.6 helix in Triton X‐100 micelles is higher than that in solution, which is indicative of difference in the Trp side chains conformation.…”

Membrane Organization and Dynamics of “Inner Pair” and “Outer Pair” Tryptophan Residues in Gramicidin Channels

“…The assignments of the conformational preferences in the populations, as deduced in DMPC from CD spectra and size-exclusion chromatograms, 38 are confirmed by the CD spectra of the same analogs in POPC bilayer (Figure 3). The fluorescence lifetimes are furthermore consistent with the conformational preferences, as retaining the inner tryptophans in the analog (Phe 13,15 gA), maintains a relatively homogeneous lifetime distribution (Figure 4b).…”

“…These results correlate well with recent single-channel and solid-state NMR results of gramicidin channels in which the inner or outer pair of tryptophan residues has been either methylated 37 or replaced with phenylalanine. 38 …”