Heavy‐Atom Labeled Transmembrane β‐Peptides: Synthesis, CD‐Spectroscopy, and X‐ray Diffraction Studies in Model Lipid Multilayer

Abstract

Transmembrane β-peptides are promising candidates for the design of well-controlled membrane anchors in lipid membranes. Here, we present the synthesis of transmembrane β-peptides with and without tryptophan anchors, as well as a novel iodine-labeled d-β(3) -amino acid. By using one or more of the heavy-atom labeled amino acids as markers, the orientation of the helical peptide was inferred based on the electron-density profile determined by X-ray reflectivity. The β-peptides were synthesized through manual Fmoc-based solid-phase peptide synthesis (SPPS) and reconstituted in unilamellar vesicles forming a right-handed 314 -helix secondary structure, as shown by circular dichroism spectroscopy. We then integrated the β-peptide into solid-supported membrane stacks and carried out X-ray reflectivity and grazing incidence small-angle X-ray scattering to determine the β-peptide orientation and its effect on the membrane bilayers. These β-peptides adopt a well-ordered transmembrane motif in the solid-supported model membrane, maintaining the basic structure of the original bilayer with some distinct alterations. Notably, the helical tilt angle, which accommodates the positive hydrophobic mismatch, induces a tilt of the acyl chains. The tilted chains, in turn, lead to a membrane thinning effect.

Semi‐Rigid Nitroxide Spin Label for Long‐Range EPR Distance Measurements of Lipid Bilayer Embedded β‐Peptides

“…Trp is known to serve as membrane anchor and orientates proteins in the lipid bilayer . Recently, it was demonstrated that this peptide motif is incorporated into a lipid bilayer preserving a well‐defined and conformationally stable 3 14 ‐helical structure . As was indicated by FRET experiments, this type of peptide has a low tendency to aggregate .…”

“…According to the basic structural parameter of an ideal 3 14 ‐helix, which is three amino acids per turn with a pitch of 0.5 nm, the hydrophobic stretch of the peptides matches the hydrophobic thickness of POPC (2 D C ≈2.71 nm) and DOPC (2 D C ≈2.68 nm) . The CD spectra recorded in solution and lipid bilayer show the typical pattern of a right‐handed 3 14 ‐helix (Figure B, left and Figures S3 and S4 in the Supporting Information) with a minimum at 195 nm, a zero‐crossing at 202 nm and a maximum at 210 nm . A small shoulder around 225 nm results from absorption of the four hTrp .…”

“…Analysis of each trace results in a broadened but, apart from P2 , still single‐peak distance distribution (Table S3 in the Supporting Information). The peptides’ structure is known to be very stable in the lipid environment . However, interactions with the lipid bilayer can increase the label′s conformational space resulting in broadening of the resulting distance distributions.…”

X-ray reflectivity reveals ionic structure at liquid crystal–aqueous interfaces

“…Neutron reflectivity contrast can be modified for the aqueous subphase 17 by changing the ra-tio of hydrogenated and deuterated water, while the amphiphile contrast can also be varied through deuteration. X-ray reflectivity (XRR) is sensitive to effective electron density, so amphiphiles can be labelled with heavy atoms to increase contrast 18 . More generally X-ray reflectivity can also be used to detect ion localisation at interfaces [19][20][21][22] .…”

Membrane‐Associated Nucleobase‐Functionalized β‐Peptides (β‐PNAs) Affecting Membrane Support and Lipid Composition

“…In case of membrane insertion, the CD signal would have been shifted to shorter wavelengths. [22] In further experiments, the β-peptide membrane interaction will be supported by atomic force microscopy (AFM), which provides more information about the distance between β-peptides and the lipid bilayer.…”