The structure of an integral membrane peptide: a deuterium NMR study of gramicidin

Abstract

Solid state deuterium NMR was employed on oriented multilamellar dispersions consisting of 1,2-dilauryl-sn-glycero-3-phosphatidylcholine and deuterium (2H) exchange-labeled gramicidin D, at a lipid to protein molar ratio (L/P) of 15:1, in order to study the dynamic structure of the channel conformation of gramicidin in a liquid crystalline phase. The corresponding spectra were used to discriminate between several structural models for the channel structure of gramicidin (based on the left- and right-handed beta 6.3 LD helix) and other models based on a structure obtained from high resolution NMR. The oriented spectrum is complicated by the fact that many of the doublets, corresponding to the 20 exchangeable sites, partially overlap. Furthermore, the asymmetry parameter, eta, of the electric field gradient tensor of the amide deuterons is large (approximately 0.2) and many of the amide groups are involved in hydrogen bonding, which is known to affect the quadrupole coupling constant. In order to account for these complications in simulating the spectra in the fast motional regime, an ab initio program called Gaussian 90 was employed, which permitted us to calculate, by quantum mechanical means, the complete electric field gradient tensor for each residue in gramicidin (using two structural models). Our results indicated that the left-handed helical models were inconsistent with our observed spectra, whereas a model based on the high-resolution structure derived by Arseniev and coworkers, but relaxed by a simple energy minimization procedure, was consistent with our observed spectra. The molecular order parameter was then estimated from the motional narrowing assuming the relaxed (right-handed) Arseniev structure. Our resultant order parameter of SZZ = 0.91 translates into an rms angle of 14 degrees, formed by the helix axis and the local bilayer normal. The strong resemblance between our spectra (and also those reported for gramicidin in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) multilayers) and the spectra of the same peptide incorporated in a lyotropic nematic phase, suggests that the lyotropic nematic phase simulates the local environment of the lipid bilayer.

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.…”

Macroscopic Orientation of Natural and Model Membranes for Structural Studies

“…The pulse spacing was 50 ¿¿s and the width of the 90° pulses were 5 ¿¿s for an 8 (39) showed that rhodopsin itself is highly ordered in these membranes and is still functionally intact. In contrast to X-ray and 31P NMR methods, these optical techniques directly probe the orientation of the protein, whereas X-ray and 31P NMR techniques determine the orientation of the lipids.…”

Deuterium Quadrupole-Coupling and Chemical-Shielding Tensors in the Model Dipeptide Glycylglycine Monohydrochloride Monohydrate

“…13 C and 15 N chemical-shift tensors for this dipeptide (14,15) As examples, the technique has been used to study biopolyhave been widely used (16,17) as models for the peptide mers such as collagen (1), membrane proteins such as grambond in biological molecules. Furthermore, the glycylglycine icidin (2,3), and synthetic polymers such as nylon (4) and dipeptide repeat is a common motif in the dragline silk from Kevlar (5). Its great utility in the study of both structure and Nephila clavipes (18), a biopolymer currently under study dynamics arises from the quadrupolar nature of the deuteron.…”