Polar Groups in Membrane Channels: Consequences of Replacing Alanines with Serines in Membrane-Spanning Gramicidin Channels
by Daily, Anna E.; Kim, Jung H.; Greathouse, Denise V.; Andersen, Olaf S.; Koeppe II, R. Erdman
To explore the consequences of burying polar, hydrogen-bonding hydroxyl groups within the hydrocarbon core of lipid bilayer membranes, we examined the structural and functional effects of alanine-to-serine substitutions in bilayer-spanning gramicidin channels. A native Ala was replaced by Ser at position 3 or 5 in the gramicidin A (gA) sequence: formyl-VG(2)A(3)LA(5)VVVWLWLWLW-ethanolamide (D-residues underlined). In the head-to-head dirners that form the conducting, membrane-spanning gA channels, these sequence positions are located near the lipid bilayer center (and subunit interface). The sequence substitutions at positions 3 and 5 were tested within the context of having either Gly or D-Ala at position 2, because D-Ala(2) causes the channel lifetimes to increase 3-fold relative to G1y2 [Mattice et al. (1995) Biochemistry 34, 6827]. Size-exclusion chromatograms and circular dichroism spectra show that the Ala -> Ser replacements are well tolerated and have little effect on channel structure. In planar bilayers, the Ser-substituted gramicidins form well-defined channels, with cation conductances that are similar to 60% of those of the reference channels. The Sersubstituted channels are structurally equivalent to native gramicidin channels, as demonstrated by the formation of heterodimeric channels between a Ser-containing subunit and a native gramicidin subunit. These hybrid channels exhibit rectification, attributable to asymmetric placement of the single Ser hydroxyl group with respect to the bilayer center. Compared to the corresponding Ala-containing reference channels, the polar Ser residues decrease the analogues' channel-forming potency by 3 orders of magnitude, indicating a substantial energetic penalty (similar to 15 kJ/mol) for burying the polar Ser side chain in the bilayer hydrophobic core.
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- 1520-4995; 0006-2960