Neighboring aliphatic/aromatic side chain interactions between residues 9 and 10 in gramicidin channels
by Koeppe II, R. Erdman; Hatchett, Jesse; Jude, Anthony Ray; Providence, Lyndon L.; Andersen, Olaf S.; Greathouse, Denise V.
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 Trp(9) was ring-labeled and D-Leu(10) 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 beta(6.3)-helical conformation that is observed for gA. H-2 NMR spectra of oriented samples in DMPC show large changes for the Trp(9) ring when residue 10 is modified, suggesting a steric interaction between D-Leu(10) and Trp(9), in agreement with previous acylation studies (R. E. Koeppe II et al. (1995) Biochemistry 34, 9299-9307). The outer quadrupolar splitting for Trp(9) is unchanged with D-Phe(10), at similar to 153 kHz, but increases by similar to 25 kHz with D-Val(10) and decreases by similar to 10 kHz with D-Ala(10). With D-Ala(10) or D-Val(10), the outer resonance splits into two in a temperature-dependent manner. The NMR spectra indicate that the side chain torsion angles chi 1 and chi 2 for Trp(9) change when residue 10 is substituted. The changes in chi 1 are small, in all cases less than 10 degrees, as is Delta chi 2 when D-Ala(10) is introduced, but with D-Val(10) and D-Phe(10) Delta chi 2 is at least 25 degrees. We conclude that D-Leu(10) helps to stabilize an optimal orientation of Trp(9) in gA channels in lipid bilayers and that changes in Trp orientation alter channel conductance and lifetime without affecting the basic channel fold.
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