Molecular Recognition at the Membrane−Water Interface: Controlling Integral Peptide Helices by Off-Membrane Nucleobase Pairing

Abstract

The aggregation and organization of membrane proteins and transmembrane peptides is related to the interacting molecular species itself and strongly depends on the lipid environment. Because of the complexity and dynamics of these interactions, they are often hardly traceable and nearly impossible to predict. For this reason, peptide model systems are a valuable tool in studying membrane associated processes since they are synthetically accessible and can be readily modified. To control and study the aggregation of peptide transmembrane domains (TMDs) the interacting interfaces of the TMDs themselves can be altered. A second less extensively studied approach targets the TMD assembly by using interaction and recognition of domains at the membrane outside as frequently found in the membrane protein interplay and protein assembly. In the present study, double helical transmembrane domains were designed and synthesized on the basis of a recently reported d,l-alternating peptide pore motif derived from gramicidin A. The highly hydrophobic and aromatic transmembrane peptide was covalently functionalized with a short peptide nucleic acid (PNA) used as specific outer-membrane recognition unit. The PNA sequences were chosen with high polarity to ensure localization within the aqueous phase. To estimate the impact of the membrane adjacent recognition on the TMD assembly by Förster resonance energy transfer (FRET), fluorescence probes were covalently attached to the side chains of the membrane spanning peptide helices. Dimerization of the TMD-peptide/PNA conjugates within unilamellar lipid vesicles was observed. The dimer/monomer ratio of TMDs can be controlled by temperature variation.

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

“…Therefore, this motif holds a tunable, conformationally stable peptide structure that is open for multiple functionalization (Schneggenburger et al 2010). The d , l alternation of the peptide backbone concordant with a double-helical β -type shape is related to natural peptide antibiotics such as gramicidin A (Kelkar and Chattopadhyay 2007) or feglymycin (Bunkóczi et al 2005).…”

“…2a, b) and the covalently linked hairpin species H-( Phe -Tyr) 5 - Trp -Trp-Gly- Lys - Pro -Gly-( Phe -Tyr) 5 Trp -Trp-OH ( 2 ) and H- Lys (NBD)-Tyr-( Phe -Tyr) 4 - Trp -Trp-Gly- Lys -([C 2 H 4 O] 2 -CH 2 -CO-gcgtgg-Lys-Lys)- Pro -Gly-( Phe -Tyr) 5 - Trp -Trp-OH ( 3 ) (Fig. 2a) (note: gcgtgg represents the nucleobase sequence of aminoethylglycine peptide nucleic acid monomers) were derived via synthetic modification applying solid-phase peptide synthesis (SPPS) and loop design (Schneggenburger et al 2010). The transmembrane helices of the latter structures ( 1 – 3 ) are well adapted to the membrane environment as optimized by sequence variation in length and composition (Küsel et al 2007).…”

“…2c, v–vi; Schneggenburger 2010). The attachment of fluorescence probes and peptide nucleic acid (PNA) recognition moieties led to functional constructs, such as compound 3 , that have already been tested for their dynamic dimerization within lipid bilayer structures via Förster resonance energy transfer (FRET) assays (Schneggenburger et al 2010). …”

Transmembrane Domain Peptide/Peptide Nucleic Acid Hybrid as a Model of a SNARE Protein in Vesicle Fusion

“…[19] In the first assay the efficiency of lipid mixing was determined by using a standard dequenching assay based on fluorescence resonance energy transfer (FRET). [20] Vesicles containing PNA1-SybTMD were prepared with nitrobenzofuran (NBD) incorporated as a donor dye and lissamine rhodamine (Rh) as an acceptor dye, both of which were attached to the head group of the 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) lipid.…”

Hybride aus Transmembranpeptiden und Peptidnucleinsäuren als Modellsystem für SNARE‐Protein‐vermittelte Vesikelfusion

“…[19] In einem ersten Ansatz wurde die Effizienz des Lipid Mixings über einen Fluoreszenz-Dequenching-Assay bestimmt, der auf resonantem Fluoreszenzenergietransfer (FRET) basiert. [20] PNA1-SybTMD enthaltende Vesikel wurden mit Nitrobenzofuran (NBD) als Donorfluorophor und Lissaminrhodamin (Rh) als Akzeptorfluorophor versehen, indem die Farbstoffe mit der Kopfgruppe von 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamin(DOPE)-Lipidmolekülen verknüpft waren.…”