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. 2005 Jan;88(1):224-34.
doi: 10.1529/biophysj.104.047456. Epub 2004 Oct 22.

Effects of phenylalanine substitutions in gramicidin A on the kinetics of channel formation in vesicles and channel structure in SDS micelles

J B Jordan  1 , P L Easton, J F Hinton
Affiliations

Effects of phenylalanine substitutions in gramicidin A on the kinetics of channel formation in vesicles and channel structure in SDS micelles

J B Jordan et al. Biophys J. 2005 Jan.

Abstract

The common occurrence of Trp residues at the aqueous-lipid interface region of transmembrane channels is thought to be indicative of its importance for insertion and stabilization of the channel in membranes. To further investigate the effects of Trp-->Phe substitution on the structure and function of the gramicidin channel, four analogs of gramicidin A have been synthesized in which the tryptophan residues at positions 9, 11, 13, and 15 are sequentially replaced with phenylalanine. The three-dimensional structure of each viable analog has been determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. These phenylalanine analogs adopt a homodimer motif, consisting of two beta6.3 helices joined by six hydrogen bonds at their NH2-termini. The replacement of the tryptophan residues does not have a significant effect on the backbone structure of the channels when compared to native gramicidin A, and only small effects are seen on side-chain conformations. Single-channel conductance measurements have shown that the conductance and lifetime of the channels are significantly affected by the replacement of the tryptophan residues (Wallace, 2000; Becker et al., 1991). The variation in conductance appears to be caused by the sequential removal of a tryptophan dipole, thereby removing the ion-dipole interaction at the channel entrance and at the ion binding site. Channel lifetime variations appear to be related to changing side chain-lipid interactions. This is supported by data relating to transport and incorporation kinetics.

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Figures

FIGURE 1
FIGURE 1
Backbone indole-NH region of the NOESY spectra of (A) native gA and (B) Phe11-gA.
FIGURE 2
FIGURE 2
Backbone indole-NH region of the NOESY spectra of (A) Phe13-gA and (B) Phe15-gA.
FIGURE 3
FIGURE 3
Minimized average structures of gA and its (left to right) Phe11-, Phe13-, and Phe15- analogs. The site of substitution is highlighted in green.
FIGURE 4
FIGURE 4
Superposition plots of the 10 best conformers of gramicidin A (A) and Phe11-gA (B) as calculated using Aria 1.2.
FIGURE 5
FIGURE 5
Superposition plots of the 10 best conformers of Phe13-gA (A) and Phe15-gA (B) as calculated using Aria 1.2.
FIGURE 6
FIGURE 6
Transverse sliced overlay plots of Phe analogs with native gramicidin A. Panels A, B, and C show Phe11-gA, Phe13-gA, and Phe15-gA, respectively, in blue overlaid on native gA in red. The site of replacement in the Phe analogs is highlighted in green.
See this image and copyright information in PMC

References

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