ORIENTATIONS OF THE TRYPTOPHAN 9 AND 11 SIDE-CHAINS OF THE GRAMICIDINCHANNEL BASED ON DEUTERIUM NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Deuterium nuclear magnetic resonance spectroscopy was used to investig ate the orientations of the indole rings of Trp(9) and Trp(11) in spec ific indole-d(5)-labeled samples of gramicidin A incorporated into dim yristoyl phosphatidylcholine bilayers in the beta(6.3) channel conform ation. The magnitudes and signs of the deuterium quadrupolar splitting s were fit to the rings and assigned to specific ring bonds, using a f ull 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 a ssignments are in close agreement with four sets of (chi(1), chi(2)) a ngles for each Trp in which the indole N-H is oriented toward the memb rane's exterior surface. (Four additional sets of (chi 1, chi(2)) angl es with the N-H's pointing toward the membrane interior are inconsiste nt with previous observations.) One of the sets of (chi(1), chi(2)) an gles for each Trp is consistent with the corresponding Trp orientation found by Arsen'ev et al. (1986. Biol. Membr. 3:1077-1104) for gramici din in sodium dodecyl sulfate micelles. Together, the H-1 and H-2 nucl ear magnetic resonance methods suggest that the Trp(9) and Trp(11) sid e chain orientations could be very similar in dimyristoyl phosphatidyl choline 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 quad rupolar splittings for ring 9 are reduced by approximately 14% due to motional averaging. Such a difference in motional averaging between ri ngs 11 and 9 is also consistent with the N-15 data of Hu et al. (1993. Biochemistry. 32:7035-7047).