Peptides derived from α-lactalbumin membrane binding helices oligomerize in presence of lipids and disrupt bilayers

Abstract

Helix A and -C of α-lactalbumin, a loosely folded amphitropic protein, perturb lipid monolayers by the formation of amyloid pore-like structures. To investigate whether these helices are able to disrupt fully formed bilayers, we designed peptides comprised of Helix A and -C, and investigated their membrane-perturbing properties. The peptides, designated A-Cage-C and A-Lnk-C, were prepared with tryptophan sites in the helical and the spacer segments in order to monitor which part were involved in membrane association under given conditions. The peptides associate with and disrupt negatively charged bilayers in a pH-dependent manner and α-helical tendencies increased upon membrane association. Both helices and the spacer segment were involved in membrane binding in the case of A-Lnk-C, and there are indications that the two helixes act in synergy to affect the membrane. However, the helices and the spacer segment could not intercalate when present as A-Cage-C at neutral conditions. At acidic pH, both helices could intercalate, but not the central spacer segment. AFM performed on bilayers under aqueous conditions revealed oligomers formed by the peptides. The presence of bilayers and acidic pHs were both drivers for the formation of these, suggestive of models for peptide oligomerization where segments of the peptide are stacked in an electrostatically favorable manner by the surface. Of the two peptides, A-Lnk-C was the more prolific oligomerizer, and also formed amyloid-fibril like structures at acidic pH and elevated concentrations. Our results suggest the peptides perturb membranes not through pore-like structures, but possibly by a thinning mechanism.

Spectroscopic and AFM characterization of polypeptide-surface interactions: Controls and lipid quantitative analyses

“…A-Lnk-C (EQLTKAEVFRELKDLKGYVGRAWKAGVISAAKFLDDDLTDDIMAVKKILDKVG) and A-Cage-C (EQLTKAEVFRELKDLNLYIQWLKDGGPSSGRPPPSFLDDDLTDDIMAVKKILDKVG) were obtained using recombinant expression in E. coli as described in [1]. Cage, based on a Trp-Cage fold (NLYIQWLKDGGPSSGRPPPS, molecular weight 2169 Da, theoretical pI=8.59, [3], and Lnk, based on a random basic sequence (KGYVGRAWKAGVISAAK, molecular weight = 1762 Da, theoretical pI=10.46), were produced by CPC Scientific using solid-phase Fmoc/t-Boc synthesis.…”

“…Handegård, M.L. Govasli, H. Wen, Ø. Halskau, 2017) [1]. In protein and polypeptide-membrane interaction studies, negatively charged lipids are often used as they are a known driver for membrane interaction.…”

Investigating the Disordered and Membrane-Active Peptide A-Cage-C Using Conformational Ensembles

“…These helices showed increased protection from solvent exchange compared to native and molten-globule controls when the protein was bound to negatively charged vesicle membranes (Figure 1A), indicating that these were key structural elements in the lipid-protein interactions [18,32]. Helix A and C, either as individual helical peptides or linked together, present interesting interfacial properties, including disruption of the bilayer through either a membrane-thinning or a pore-like mechanism associated with protein aggregation [33,34]. .…”

“…(B) Cartoon sketch of the A-Cage-C, derived from full-length ALA. Joining the two helices together with a short peptide to allow them to act together in the absence of the tertiary structure of ALA [34].…”

“…Here, we investigate the chimeric A-Cage-C polypeptide, one of the artificially designed recombinant peptides previously investigated by Strømland et al [34]. A-Cage-C joins the A and C helices of bovine α-Lactalbumin (BLA) using the Tryptophan Cage, a small folding unit (Figure 1B) [35].…”