Received September 2, 1999

Revised Manuscript Received December 3, 1999

Abstract:

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 ^6.3-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 1 and 2 for Trp⁹ change when residue 10 is substituted. The changes in 1 are small, in all cases less than 10, as is 2 when D-Ala¹⁰ is introduced, but with D-Val¹⁰ and D-Phe¹⁰ 2 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.

Download the full text: PDF | HTML