Orientations of the Tryptophan 9 and 11 Side-Chains of the Gramicidin Channel Based on Deuterium Nuclear-Magnetic-Resonance Spectroscopy

by Koeppe, R. E.; Killian, J. A.; Greathouse, D. V.

Deuterium nuclear magnetic resonance spectroscopy was used to investigate the orientations of the indole rings of Trp(9) and Trp(11) in specific indole-d(5)-labeled samples of gramicidin A incorporated into dimyristoyl phosphatidylcholine bilayers in the beta(6.3) channel conformation. The magnitudes and signs of the deuterium quadrupolar splittings were fit to the rings and assigned to specific ring bonds, using a full rotation search of the chi(1) and chi(2) angles of each Trp and a least-squares method. Unique assignments were obtained. The data and assignments are in close agreement with four sets of (chi(1), chi(2)) angles for each Trp in which the indole N-H is oriented toward the membrane's exterior surface. (Four additional sets of (chi 1, chi(2)) angles with the N-H's pointing toward the membrane interior are inconsistent with previous observations.) One of the sets of (chi(1), chi(2)) angles for each Trp is consistent with the corresponding Trp orientation found by Arsen'ev et al. (1986. Biol. Membr. 3:1077-1104) for gramicidin in sodium dodecyl sulfate micelles. Together, the H-1 and H-2 nuclear magnetic resonance methods suggest that the Trp(9) and Trp(11) side chain orientations could be very similar in dimyristoyl phosphatidylcholine membranes and in sodium dodecyl sulfate micelles. The data for Trp(11) could be fit using a static quadrupolar coupling constant of 180 kHz under the assumption that the ring is essentially immobile. By contrast, Trp(9) could be fit only under the assumption that the quadrupolar splittings for ring 9 are reduced by approximately 14% due to motional averaging. Such a difference in motional averaging between rings 11 and 9 is also consistent with the N-15 data of Hu et al. (1993. Biochemistry. 32:7035-7047).

Biophysical Journal
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1542-0086; 0006-3495