title: Polar groups in membrane channels: consequences of replacing alanines with serines in membrane-spanning gramicidin channels.
abstract: 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 dimers 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 Gly(2) [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 approximately 60% of those of the reference channels. The Ser-substituted 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 ( approximately 15 kJ/mol) for burying the polar Ser side chain in the bilayer hydrophobic core.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
authors.forename: Anna E
authors.initials: AE
authors.lastname: Daily
authors.forename: Jung H
authors.initials: JH
authors.lastname: Kim
authors.forename: Denise V
authors.initials: DV
authors.lastname: Greathouse
authors.forename: Olaf S
authors.initials: OS
authors.lastname: Andersen
authors.forename: Roger E
authors.initials: RE
authors.lastname: Koeppe
mesh_terms: D000409:Alanine / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004594:Electrophysiology; D006096:Gramicidin / Q000378:metabolism*; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D012694:Serine / Q000737:chemistry*
chemical_list: D007473:Ion Channels; D006096:Gramicidin; D012694:Serine; D000409:Alanine