Tilt Angles of Transmembrane Model Peptides in Oriented and Non-Oriented Lipid Bilayers as Determined by 2H Solid-State NMR

Abstract

Solid-state NMR methods employing (2)H NMR and geometric analysis of labeled alanines (GALA) were used to study the structure and orientation of the transmembrane alpha-helical peptide acetyl-GWW(LA)(8)LWWA-amide (WALP23) in phosphatidylcholine (PC) bilayers of varying thickness. In all lipids the peptide was found to adopt a transmembrane alpha-helical conformation. A small tilt angle of 4.5 degrees was observed in di-18:1-PC, which has a hydrophobic bilayer thickness that approximately matches the hydrophobic length of the peptide. This tilt angle increased slightly but systematically with increasing positive mismatch to 8.2 degrees in di-C12:0-PC, the shortest lipid used. This small increase in tilt angle is insufficient to significantly change the effective hydrophobic length of the peptide and thereby to compensate for the increasing hydrophobic mismatch, suggesting that tilt of these peptides in a lipid bilayer is energetically unfavorable. The tilt and also the orientation around the peptide axis were found to be very similar to the values previously reported for a shorter WALP19 peptide (GWW(LA)(6)LWWA). As also observed in this previous study, the peptide rotates rapidly around the bilayer normal, but not around its helix axis. Here we show that these properties allow application of the GALA method not only to macroscopically aligned samples but also to randomly oriented samples, which has important practical advantages. A minimum of four labeled alanine residues in the hydrophobic transmembrane sequence was found to be required to obtain accurate tilt values using the GALA method.

Influence of Flanking Residues on Tilt and Rotation Angles of Transmembrane Peptides in Lipid Bilayers. A Solid-State 2H NMR Study

“…RMSD values of WALP23 increase with thinner bilayers as observed previously (14). In the case of KALP23, RMSD values are rather small for all bilayer thicknesses, indicating a good fit.…”

“…This method, called geometric analysis of labeled alanines (GALA), allows for very accurate determination of tilt angles of WALP peptides in either oriented or nonoriented PC bilayers (11,14). In addition, the direction in which the peptides are tilted, as determined by their 1 Abbreviations: PC, phosphatidylcholine; NMR, nuclear magnetic resonance; TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; tBu, tert-butyl; d 4-Ala, deuterated L-alanine-d4; Fmoc, 9-fluorenylmethyloxycarbonyl; di-C12:0-PC, 1,2-dilauroyl-sn-glycero-3-phosphocholine; di-C13:0-PC, 1,2-ditridecanoyl-sn-glycero-3-phosphocholine; di-C14: 0-PC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; di-C18:1-PC, 1,2-dioleoyl-sn-glycero-3-phosphocholine.…”

“…In all cases the spectra were similar to those reported previously for WALP/DMPC samples (6), confirming a bilayer organization of the lipids (data not shown). 2 H NMR experiments were performed at 76.78 MHz using a quadrupolar echo sequence as described previously (14).…”

“…Table 2 shows that for almost all labeled positions systematic changes in quadrupolar splitting occur, whereby the quadrupolar splitting decreases with increasing bilayer thickness from di-12:0-PC to di-18:1-PC. From the quadrupolar splittings obtained for the different labeled positions, tilt and rotation angles were calculated on the basis of an R-helical geometry of the peptides and on a geometric analysis of labeled alanine (GALA) (14). The results are shown in Table 3.…”

“…In particular, the so-called WALP peptides have been used as tools to monitor the consequence of hydrophobic mismatch for the organization of peptides and lipids in model membranes (reviewed in ref 15). WALP peptides are a family of R-helical transmembrane model peptides made of a central leucine alanine stretch that is flanked by two tryptophan residues on each side.Recently, a technique based on solid-state 2 H NMR using deuterated alanines has been developed to analyze structural properties of transmembrane peptides in model membranes (10,11,14,16). This method, called geometric analysis of labeled alanines (GALA), allows for very accurate determination of tilt angles of WALP peptides in either oriented or…”