Membrane organization and dynamics of "inner pair" and "outer pair" tryptophan residues in gramicidin channels

Abstract

The linear ion channel peptide gramicidin serves as an excellent prototype for monitoring the organization, dynamics, and function of membrane-spanning channels. The tryptophan residues in gramicidin channels are crucial for establishing and maintaining the structure and function of the channel in the membrane bilayer. In order to address the basis of differential importance of tryptophan residues in the gramicidin channel, we monitored the effects of pairwise substitution of two of the four gramicidin tryptophans, the inner pair (Trp-9 and -11) and the outer pair (Trp-13 and -15), using a combination of steady state and time-resolved fluorescence approaches and circular dichroism spectroscopy. We show here that these double tryptophan gramicidin analogues adopt different conformations in membranes, suggesting that the conformational preference of double tryptophan gramicidin analogues is dictated by the positions of the tryptophans in the sequence. These results assume significance in the context of recent observations that the inner pair of tryptophans (Trp-9 and -11) is more important for gramicidin channel formation and channel conductance. These results could be potentially useful in analyzing the effect of tryptophan substitution on the functioning of ion channels and membrane proteins.

FIGURE 1

Amino acid sequence of gramicidin A and the double tryptophan analogs used. Alternating d-amino acid residues are underlined. Aromatic amino acids are highlighted and their positions shown. Tryptophan residues at positions 9 and 11 are designated as the ‘inner pair’ while the tryptophan residues at positions 13 and 15 are designated as the ‘outer pair’. The analog in which the inner pair of tryptophans are substituted by phenylalanine is denoted as Phe^,gA, while the analog in which the outer pair of tryptophans are substituted is termed Phe^,gA.

FIGURE 2

(a) Fluorescence emission spectra of gramicidin analogs in vesicles of POPC. The spectra corresponding to Phe^,gA (—) and Phe^,gA (---) are shown. The excitation wavelength was 280 nm in both cases. Spectra are intensity-normalized at the respective emission maximum. (b) Effect of changing excitation wavelength on the wavelength of maximum emission for Phe^,gA (■) and Phe^,gA (•). Lines joining data points are provided merely as viewing guides. The ratio of peptide to POPC was 1:50 (mol/mol) and the concentration of POPC was 0.85 mM. See Experimental Section for more details.

FIGURE 3

Far-UV CD spectra of gramicidin (— - —), Phe^,gA (—) and Phe^,gA (---) in POPC vesicles. The ratio of peptide to POPC was 1:50 (mol/mol) and the concentration of POPC was 0.85 mM. See Experimental Section for other details.

FIGURE 4

MEM lifetime distribution of tryptophan residues in (a) Phe^,gA (---) and (b) Phe^,gA (—) in POPC vesicles. The reduced χ² ratios for the distributions are 0.97 and 1.06, respectively. The ratio of peptide to POPC was 1:50 (mol/mol) and the concentration of POPC was 0.85 mM. See Experimental Section for other details.

FIGURE 5

Time-resolved fluorescence anisotropy decay of tryptophan residues in Phe^,gA (—) and Phe^,gA (---) in vesicles of POPC. The excitation wavelength was 295 nm and emission was monitored at 340 nm with a combination of a monochromator and a 320 nm cut-off filter, using a TCSPC setup. Inset shows the relative magnitudes of ϕ₂. The ratio of peptide to POPC was 1:50 (mol/mol) and the concentration of POPC was 0.85 mM. See Experimental Section for other details.

FIGURE 6

A schematic representation of the effect of pair-wise substitution of tryptophan residues of the gramicidin channel. Our results suggest that the double tryptophan gramicidin analog Phe^,gA with ‘outer pair’ tryptophan residues (Trp -13 and -15) assumes a conformation which is more non-channel-like. On the other hand, the analog Phe^,gA with ‘inner pair’ of tryptophans (Trp-9 and -11) assumes a channel-like conformation. See Figure 1 and text for details.