Deuterium NMR of Val1.cntdot..cntdot..cntdot.(2-2H)Ala3.cntdot..cntdot..cntdot.gramicidin A in oriented DMPC bilayers

Abstract

Deuterium NMR is used to study the selectively labeled Val1...(2-2H)Ala3...gramicidin A molecule to investigate the structure and dynamics of the C alpha-2H bond in the Ala3 residue of gramicidin. Val1...(2-2H)Ala3...gramicidin A is synthesized, purified, and characterized and then incorporated into oriented bilayers of dimyristoylphosphatidylcholine sandwiched between glass coverslips. Phosphorus NMR line shapes obtained from this sample are consistent with the presence of the bilayer phase and indicate that no nonbilayer phases are present in significant amounts. Deuterium NMR line shapes obtained from this sample indicate that the motional axis of the gramicidin Ala3 residue is parallel to the coverslip normal, that the distribution of motional axis orientations has a width of 2 degrees, and that only one major conformational and dynamical state of the Ala3 C alpha-2H bond is observed on the NMR time scale. Furthermore, the Ala3 C alpha-2H bond angle relative to the motional axis is 19-20 degrees if fast axial rotation is assumed to be the only motion present but is less than or equal to 19-20 degrees in the absence of such an assumption. This result indicates that various double-stranded, helical dimer models are very unlikely to represent the structure of gramicidin in the sample studied but that the single-stranded, beta 6.3 helical dimer models are consistent with the experimental data. However, a definitive distinction between the left-handed, single-stranded, beta 6.3 helical dimer model and the right-handed, single-stranded, beta 6.3 helical dimer model cannot be made on the basis of the experimental data obtained in this study.

NMR order parameter analysis of a peptide plane aligned in a lyotropic liquid crystal

“…Arseniev et al [47] first determined that the helix was right handed using 2D NMR studies of gramicidin A in lipid micelles. The helical sense of the gramicidin in phospholipid bilayers was determined to be right handed by the use of additional specific 15N labelling [48], a result later confirmed by the 2H experiments discussed above [41][42][43]. For the amide nitrogen shielding tensor of Ala3, a33 was determined to lie ~ 14 5= 2 ~ off the N-H bond using 13C-15N dipolar coupled powder spectra [48,49].…”

“…Assuming fast axial rotation of the long molecular axis of the gramicidin parallel to the membrane normal, this is consistent with the angle between the N-2H vector along the backbone and the molecular axis of rotation being equal to 16 ~ Davis [40] incorporated exchange-labelled gramicidin in an oriented lipid phase and obtained a value of 164-7 ~ for this angle, with a splitting of ~ 123 kHz. Hing et al [41,42] used gramicidin specifically deuterated at Ala3 C~-2H aligned in phospholipid bilayers and obtained results indicating that the motional axis is parallel to the bilayer normal, and if fast axial reorientation is the only motion present, then the gramicidin is a right handed helix. The handedness of the helix was confirmed by Prosser et al [43] for gramicidin A in unoriented phospholipid multilayers using additional C~-2H labels.…”

Solid-State NMR Studies of a Diverged Microsomal Amino-Proximate Δ12 Desaturase Peptide Reveal Causes of Stability in Bilayer: Tyrosine Anchoring and Arginine Snorkeling

“…Upon addition of Yb 31 to the bicelles, the quadrupolar splitting doubled from 15 kHz to 30 kHz. The doubling of the quadrupolar splitting has been observed when the phospholipids' acyl chains are deuterated in magnetically aligned phospholipid bilayers utilizing DMPC d54 (54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67). This doubling of the quadrupolar splitting indicates that the TM-A peptide rotates rapidly about the bilayer normal (45).…”

Solid-State NMR Structural Determination of Components in an Ion Channel Switch Biosensor

“…Solid-state NMR studies of labelled gA derivatives in macroscopically aligned multilayers have yielded much information on the structure and dynamics of the gA backbone. [1][2][3][4][5][6][7][8][9][10][11] With alignment, sharp resonance lines can be obtained for each labelled site in the molecule, the positions of which are determined by the average orientation of the site with respect to the magnetic field. By using oriented gA molecules, the structure of gA in BLMs and ion binding sites [12,13] were determined and a better understanding of the gA ion channel function in different chain length BLMs was obtained.…”