A Novel Heavy‐Atom Label for Side‐Specific Peptide Iodination: Synthesis, Membrane Incorporation and X‐ray Reflectivity

Abstract

Structural parameters, such as conformation, orientation and penetration depth of membrane-bound peptides and proteins that may function as channels, pores or biocatalysts, are of persistent interest and have to be probed in the native fluid state of a membrane. X-ray scattering in combination with heavy-atom labeling is a powerful and highly appropriate method to reveal the position of a certain amino acid residue within a lipid bilayer with respect to the membrane normal axis up to a resolution of several Angstrøm. Herein, we report the synthesis of a new iodine-labeled amino acid building block. This building block is intended for peptide incorporation to provide high intensities for electron density difference analysis of X-ray reflectivity data and improve the labeling potential for the lipid bilayer head-group and water region. The novel building block as well as the commercially available non-iodinated analogue, required for X-ray scattering, was implemented in a transmembrane peptide motif via manual solid-phase peptide synthesis (SPPS) following the fluorenylmethyloxycarbonyl (Fmoc)-strategy. The derived peptides were reconstituted in lipid vesicles as well as in highly aligned multilamellar lipid stacks and investigated via circular dichroism (CD) and X-ray reflectivity. Thereby, it has been revealed that the bulky iodine probe neither causes conformational change of the peptide structure nor lamellar disordering of the membrane complexes.

Peptide model helices in lipid membranes: insertion, positioning, and lipid response on aggregation studied by X-ray scattering

“…The distance between the maxima of the electron density difference curve, associated with the distance of the two iodine labels is about 32 Å (see the Electronic Supplementary Material). This is in good agreement with the expectation based on a peptide length of 26 Å and the additional spacing of the Phe(4-I) side chains attached at both ends of the double helix and pointing to opposite directions (Schneggenburger et al 2009). …”

“…The homodimer species 2 adopts a membrane spanning orientation in DLPC bilayers (Küsel et al 2007) and likewise tends to aggregation (Schneggenburger et al 2009). Therefore, the GID approach was undertaken to evaluate if the latter observation of a membrane insertion and transmembrane alignment is likewise true for the designed hairpin species ( 2 ) and the recognition system ( 3 ) (Fig.…”

“…With the synthesis of the novel double iodinated amino acid building block Fmoc-5,5-diiodoalyllglycine-OH, the use of an enhanced in-house reflectivity set up including substrate sizes of 15 × 25 mm and the addition of 5 mol% cholesterol to the lipid phase enabled recording up to seven lamellar orders from samples with a P/L = 1/50. The analysis of the electron density difference profiles then allowed for the determination of the iodine positions with high accuracy (Schneggenburger et al 2009). Even for the Fmoc-Phe(4-I)-OH single label, P/L ratios of 1/50 turned out to be sufficient.…”

“…Finally, we must be cautious since iodine-labeling itself may influence peptide/lipid interactions. However, there is no obvious reason why attachment of the iodine labels at different positions should cause different peptide positioning, especially as the iodine labels have been shown to have only minor effects on the peptide secondary structure (Schneggenburger et al 2009). …”

“…The phases have been extracted by the 1D swelling method of pure DLPC-lipid samples (Schneggenburger et al 2009). The electron density profile ρ ( z ) normal to the interface is computed by N 0 Fourier coefficients f n  =  I n   q z (Eq.…”