β-Glutamine-mediated self-association of transmembrane β-peptides within lipid bilayers

Abstract

Transmembrane β-peptide helices and their association in lipid membranes are still widely unexplored. We designed and synthesized transmembrane β-peptides harboring different numbers of d-β³-glutamine residues (^hGln) by solid phase peptide synthesis. By means of circular dichroism spectroscopic measurements, the secondary structure of the β-peptides reconstituted into unilamellar vesicles was determined to be similar to a right-handed 3₁₄-helix. Fluorescence spectroscopy using d-β³-tryptophan residues strongly suggested a transmembrane orientation. Two or three ^hGln served as recognition units between the helices to allow helix-helix assembly driven by hydrogen bond formation. The association state of the transmembrane β-peptides as a function of the number of ^hGln residues was investigated by fluorescence resonance energy transfer (FRET). Therefore, two fluorescence probes (NBD, TAMRA) were covalently attached to the side chains of the transmembrane β-peptide helices. The results clearly demonstrate that only β-peptides with ^hGln as recognition units assemble into oligomers, presumably trimers. Temperature dependent FRET experiments further show that the strength of the helix-helix association is a function of the number of ^hGln residues in the helix.

Fig. 1. Schematic illustration of hydrogen bond formation of d-β³-glutamine residues.

Fig. 2. Basic structure of the designed β-peptides (H-^hLys₂-^hTrp₂-^hVal₁₉-^hTrp₂-^hLys₂-NH₂).

Fig. 3. Schematic view on two 3₁₄-helical β-peptides oriented in an antiparallel fashion with three amino acids forming one turn and zero ^hGln (9/10/11, left), two ^hGln (12/13/14, middle) and three ^hGln (15/16/17, right) recognition units. The β-peptides were acetylated or labelled with NBD and TAMRA for FRET-analysis and synthesized from N- to C-terminus.

Fig. 4. CD-spectra of 9, 12 and 15 in DOPC LUVs (peptides concentration: 38 μm, P/L-ratio = 1/20, 25 °C).

Fig. 5. Fluorescence spectra of equimolar mixtures of 10/11, 13/14 and 16/17 in DOPC LUVs (peptide concentration: 12 μm, P/L-ratio = 1/500, 25 °C).

Fig. 6. (A + B) Fluorescence emission spectra of NBD-labelled β-peptides (donor, 6.0 μm) and varying amounts of TAMRA-labelled species from χ_A = 0–0.5 determined at 25 °C. The non-labelled compound was added to keep the total peptide concentration constant (12 μm) and the P/L-ratio at 1/500 (DOPC). (A) 12/13/14 with two ^hGln and (B) 15/16/17 with three ^hGln. (C) Relative changes in NBD-fluorescence emission (F/F₀) as a function of increasing acceptor concentration (χ_A) are plotted for all three cases at 25 °C (9/10/11 with zero ^hGln, 12/13/14 with two ^hGln and 15/16/17 with three ^hGln). The grey solid line is the result of a model according to Wolber et al., by taking only statistical occurrence of FRET in vesicles without the formation of aggregates into account., A monomer–dimer equilibrium does not explain the data. Even the assumption of a pure dimer (dashed black line) does not explain the observed plots. The solid lines are results of the global fit analysis, which takes a monomer–trimer equilibrium into account with K_D = (17.2 ± 7.0) × 10^–8 MF² (two ^hGln) and K_D = (4.4 ± 4.3) × 10^–8 MF² (three ^hGln).

Fig. 7. Relative changes in NBD-fluorescence emission (F/F₀) as a function of increasing acceptor concentration (χ_A) are plotted for 12/13/14 with two ^hGln (A) and 15/16/17 with three ^hGln (B) as recognition units at 25 °C and 60 °C. The grey solid line is the result of a model according to Wolber et al., taking only statistical occurrence of FRET in vesicles without the formation of aggregates into account., The black solid lines are the results of a global fit analysis which takes a monomer–trimer equilibrium into account with K_D = (17.2 ± 7.0) × 10^–8 MF² (two ^hGln) and K_D = (4.4 ± 4.3) × 10^–8 MF² (three ^hGln).