Neighboring Aliphatic/Aromatic Side Chain Interactions between Residues 9 and 10 in Gramicidin Channels

• Published in: Biochemistry (Easton) 2000-03, Vol.39 (9), p.2235-2242
• Main Authors: Koeppe, Roger E, Hatchett, Jesse, Jude, Anthony R, Providence, Lyndon L, Andersen, Olaf S, Greathouse, Denise V
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Chromatography, Gel; Circular Dichroism; Deuterium; Gramicidin - analogs & derivatives; Gramicidin - chemistry; Ion Channels - chemistry; Leucine - chemistry; Lipid Bilayers - chemistry; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylcholines - chemistry; Protein Conformation; Tryptophan - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9920679

The interactions between an aliphatic or phenyl side chain and an indole ring in a phospholipid environment were investigated by synthesizing and characterizing gramicidins in which Trp9 was ring-labeled and d-Leu10 was replaced by d-Val, d-Ala, or d-Phe. All three analogues form conducting channels, with conductances that are lower than that of gramicidin A (gA) channels. The channel lifetimes vary by less than 50% from that of gA channels. Circular dichroism spectra and size-exclusion chromatography show that the conformation of each analogue in dimyristoylphosphatidylcholine (DMPC) vesicles is similar to the right-handed β6.3-helical conformation that is observed for gA. 2H NMR spectra of oriented samples in DMPC show large changes for the Trp9 ring when residue 10 is modified, suggesting a steric interaction between d-Leu10 and Trp9, in agreement with previous acylation studies (R. E. Koeppe II et al. (1995) Biochemistry 34, 9299−9307). The outer quadrupolar splitting for Trp9 is unchanged with d-Phe10, at ∼153 kHz, but increases by ∼25 kHz with d-Val10 and decreases by ∼10 kHz with d-Ala10. With d-Ala10 or d-Val10, the outer resonance splits into two in a temperature-dependent manner. The NMR spectra indicate that the side chain torsion angles χ1 and χ2 for Trp9 change when residue 10 is substituted. The changes in χ1 are small, in all cases less than 10°, as is Δχ2 when d-Ala10 is introduced, but with d-Val10 and d-Phe10 Δχ2 is at least 25°. We conclude that d-Leu10 helps to stabilize an optimal orientation of Trp9 in gA channels in lipid bilayers and that changes in Trp orientation alter channel conductance and lifetime without affecting the basic channel fold.