Orientations of the tryptophan 9 and 11 side chains of the gramicidin channel based on deuterium nuclear magnetic resonance spectroscopy

Abstract

Deuterium nuclear magnetic resonance spectroscopy was used to investigate the orientations of the indole rings of Trp9 and Trp11 in specific indole-d5-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 1H and 2H nuclear magnetic resonance methods suggest that the Trp9 and Trp11 side chain orientations could be very similar in dimyristoyl phosphatidylcholine membranes and in sodium dodecyl sulfate micelles. The data for Trp11 could be fit using a static quadrupolar coupling constant of 180 kHz under the assumption that the ring is essentially immobile. By contrast, Trp9 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 15N data of Hu et al. (1993. Biochemistry. 32:7035-7047).

Methods for studying transmembrane peptides in bicelles: consequences of hydrophobic mismatch and peptide sequence

“…S pw is the molecular order parameter that describes any conformational instability or overall peptide wobble. Previous studies have shown that transmembrane domains have relatively high order parameters [38][39][40] and studies of membrane-associated peptides in bicelles have also revealed high order parameters [37]. Therefore, a value of 1 for S pw was used here.…”

The Preference of Tryptophan for Membrane Interfaces

“…Data Analysis-2 H NMR spectra have been used to examine the orientations of Trp indole rings in gA channels (16,(42)(43)(44). Because deuterium has a spin number of 1, each deuterium produces two symmetric peaks in a 2 H NMR measurement.…”

Is There a Preferential Interaction between Cholesterol and Tryptophan Residues in Membrane Proteins?

“…Thus, four quadrupolar splittings are expected for WALP23 with indole ring-deuterated tryptophans. Previous 2 H NMR experiments on oriented samples containing the tryptophan-flanked gramicidin A (21,45,46) or WALP peptides (47,48) showed that not all splittings are always detected for either orientation of the bilayers with respect to the magnetic field. Looking at the 2 H NMR spectra for WALP23-d 5 -Trp2, we can distinguish four splittings for the 90°orientation ( Figure 7A, right column) compared to only three splittings for the 0°o rientation ( Figure 7A, left column).…”

“…In a phospholipid bilayer, the long axis and therefore also the flat face of the complex ring system of cholesterol are oriented almost parallel to the bilayer normal (56,57). It is possible to calculate the orientation of the tryptophan side chain with respect to the bilayer from the quadrupolar splittings of a deuterated indole ring if the spectral assignments are known (21,46). Preliminary attempts to calculate the orientations of the deuterated tryptophans of WALP23-d 5 -Trp2 and WALP23-d 5 -Trp3 using an automatic assignment procedure yielded for both tryptophan positions a reliable minimum for a single orientation.…”