Gramicidin channels that have no tryptophan residues

Abstract

In order to understand how aromatic residues modulate the function of membrane-spanning proteins, we examined the role of the four tryptophans in gramicidin A (gA) in determining the average duration and permeability characteristics of membrane-spanning gramicidin channels; the tryptophan residues were replaced by tyrosine (gramicidin T, gT), tyrosine O-benzyl ether [gramicidin T(Bzl), gT(Bzl)], naphthylalanine (gramicidin N, gN), and phenylalanine (gramicidin M enantiomer, gM-). These analogues form channels with durations and conductances that differ some 10- and 16-fold, respectively. The single-channel conductance was invariably decreased by the Trp----Yyy replacement, and the relative conductance alterations were similar in phosphatidylcholine (DPhPC) and monoglyceride (GMO) bilayers. The duration variations exhibited a more complex pattern, which was quite different in the two membrane environments: in DPhPC bilayers, gN channels have an average duration that is approximately 2-fold longer than that of gA channels; in GMO bilayers, the average duration of gN channels is about one-tenth that of gA channels. The sequence-dependent alterations in channel function do not result from alterations in the channels' peptide backbone structure, because heterodimers can form between the different analogues and gramicidine A, and there is no energetic cost associated with heterodimer formation [cf. Durkin, J. T., Koeppe, R. E., II, & Andersen, O. S. (1990) J. Mol. Biol. 211, 221]. The alterations in permeability properties are consistent with the notion that Trp residues alter the energy profile for ion permeation through long-range electrostatic interactions.

Exploring membrane organization and dynamics by the wavelength-selective fluorescence approach

“…The results from the above study point out the motional restriction experienced by the tryptophans at the peptide Á/lipid interface of the gramicidin channel. This is consistent with other studies (Becker et al, 1991;Fonseca et al, 1992) in which such restrictions are thought to be imposed due to hydrogen bonding between the indole rings of the tryptophan residues in the channel conformation and the neighboring lipid carbonyls. The significance of such organization in terms of functioning of the channel is brought out by the fact that substitution, photodamage, or chemical modification of these tryptophans are known to give rise to channels with altered conformation and reduced conductivity.…”

“…Due to its aromaticity, the tryptophan residue is capable of p Á/p interactions and of weakly polar interactions (Burley and Petsko, 1988). The amphipathic character of tryptophan gives rise to its hydrogen bonding ability which could account for its orientation in membrane proteins and its function through long-range electrostatic interaction (Fonseca et al, 1992). The amphipathic character of tryptophan also explains its interfacial localization in membranes due to its tendency to be solubilized in this region of the membrane, besides favorable electrostatic interactions and hydrogen bonding.…”

The Preference of Tryptophan for Membrane Interfaces

“…7 and (27). Moreover, the [Phe 9,11,13,15 ]gA channels have a Cs ϩ conductance that is 6-fold less than that of gA channels (at 200 mV) (26), whereas the conductance of [1-Me-Trp 9,11,13,15 ]gA channels is reduced by only 25%. This comparison of 1-Me-Trp versus Phe further demonstrates the importance of the Trp dipole moment for the ion permeability of the gA channels.…”

“…1C) (22,23). Changing the membrane environment (24,25) or the amino acid sequence (18,19,26) can alter the gA folding preference, in some cases promoting the folding into double-stranded (DS) dimeric structures (Fig. 1D).…”

“…Early studies showed that substitution of any one of the four Trps by Phe decreases the gA single channel conductance (27), with the conductance decrease being more pronounced for the "inner" substitutions, at position 9 or 11 (27). When all four Trps are replaced by Phe, the resulting analogue, designated "gramicidin M," forms channels with a conductance that is 6-fold less than that of the native gA channels (26,28). Moreover, the preferred fold of gramicidin M in a lipid environment is one or more DS structures (19,29), which are not able to conduct ions (18).…”