- Polar groups in membrane channels: Consequences of replacing alanines with serines in membrane-spanning gramicidin channels
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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-VG2A 3LA5VVVWLWLWLW-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 Gly2 [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 ~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 (~15 kJ/mol) for burying the polar Ser side chain in the bilayer hydrophobic core.
- Daily, Anna E.,Kim, Jung H.,Greathouse, Denise V.,Andersen, Olaf S.,Koeppe, Roger E.
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- Discovery of gramicidin A analogues with altered activities by multidimensional screening of a one-bead-one-compound library
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Gramicidin A (1) is a peptide antibiotic that disrupts the transmembrane ion concentration gradient by forming an ion channel in a lipid bilayer. Although long used clinically, it is limited to topical application because of its strong hemolytic activity and mammalian cytotoxicity, likely arising from the common ion transport mechanism. Here we report an integrated high-throughput strategy for discovering analogues of 1 with altered biological activity profiles. The 4096 analogue structures are designed to maintain the charge-neutral, hydrophobic, and channel forming properties of 1. Synthesis of the analogues, tandem mass spectrometry sequencing, and 3 microscale screenings enable us to identify 10 representative analogues. Re-synthesis and detailed functional evaluations find that all 10 analogues share a similar ion channel function, but have different cytotoxic, hemolytic, and antibacterial activities. Our large-scale structure-activity relationship studies reveal the feasibility of developing analogues of 1 that selectively induce toxicity toward target organisms.
- Hamamoto, Hiroshi,Inoue, Masayuki,Itoh, Hiroaki,Panthee, Suresh,Paudel, Atmika,Sekimizu, Kazuhisa,Takada, Yuri
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