Formation of non-beta 6.3-helical gramicidin channels between sequence-substituted gramicidin analogues

Abstract

Using the linear gramicidins as an example, we have previously shown how the statistical properties of heterodimeric (hybrid) channels (formed between the parent [Val1]gramicidin A (gA) and a sequence-altered analogue) can be used to assess whether the analogue forms channels that are structurally equivalent to the parent channels (Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. J. Mol. Biol. 211:221-234). Generally, the gramicidins are tolerant of amino acid sequence alterations. We report here an exception. The optically reversed analogue, gramicidin M- (gM-) (Heitz, F., G. Spach, and Y. Trudelle. 1982. Biophys. J. 40:87-89), forms channels that are the mirror-image of [Val1]gA channels; gM- should thus form no hybrid channels with analogues having the same helix sense as [Val1]gA. Surprisingly, however, gM- forms hybrid channels with the shortened analogues des-Val1-[Ala2]gA and des-Val1-gC, but these channels differ fundamentally from the parent channels: (a) the appearance rate of these heterodimers is only approximately 1/10 of that predicted from the random assortment of monomers into conducting dimers, indicating the existence of an energy barrier to their formation (e.g., monomer refolding into a new channel-forming conformation); and (b), once formed, the hybrid channels are stabilized approximately 1,000-fold relative to the parent channels. The increased stability suggests a structure that is joined by many hydrogen bonds, such as one of the double-stranded helical dimers shown to be adopted by gramicidins in organic solvents (Veatch, W. R., E. T. Fossel, and E. R. Blout. 1974. Biochemistry. 13:5249-5256).

The Preference of Tryptophan for Membrane Interfaces

“…The four Trps at the C-terminal cluster at the membrane-water interface (Fig. 1C) and are important for the SS conformational preference of gA in lipid bilayers (18,19). The DS structures (Fig.…”

“…Previous experiments on gA 3 suggested that the indole dipole moment (13)(14)(15)(16)(17) has a direct influence on membrane protein function (ion permeation) and that indole NH hydrogen bonding is important for the conformational preference of gA (18,19). More generally, indole N-H hydrogen bonding appears to be important for protein folding and dynamics (20).…”

“…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).…”

“…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). These results suggest that all four Trps are important for gA structure and function, although the contribution of each Trp may differ.…”

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“…1c). The energetic cost of burying the amphipathic Trp residues will destabilize the DS channels relative to their SS counterparts 14 ; one cannot neglect the importance of the (membrane) environment for the gramicidin's conformational preference.…”