How Peptide Molecular Structure and Charge Influence the Nanostructure of Lipid Bicontinuous Cubic Mesophases: Model Synthetic WALP Peptides Provide Insights

Abstract

Nanostructured bicontinuous lipidic cubic phases are used for the encapsulation of proteins in a range of applications such as in meso crystallization of transmembrane proteins and as drug delivery vehicles. The retention of the nanoscale order of the cubic phases subsequent to protein incorporation, as well as retention of the protein structure and function, is essential for all of these applications. Herein synthetic peptides (WALP21, WALPS53, and WALPS73) with a common α-helical hydrophobic domain, but varying hydrophilic loop size, were designed to systematically examine the effect of peptide structure and charge on bicontinuous cubic phases. The effect of the cubic phases on the secondary structure of the peptides was also investigated. The incorporation of the WALP peptides in cubic phases formed by a range of lipids showed that hydrophobic mismatch of the peptides with the lipid bilayers, the hydrophilic domain size, and peptide charge were all significant factors determining the response of the lipid nanomaterial to protein insertion. As charge repulsion had the most significant effect on the phase transitions observed, we suggest that buffer pH and salt concentration must be carefully considered to ensure cubic mesophase retention. Importantly, the WALP peptides were found to have a different conformation depending on the local lipid environment. Such structural changes could potentially affect membrane protein function, which is crucial for both current and prospective applications.

Using SANS with Contrast-Matched Lipid Bicontinuous Cubic Phases To Determine the Location of Encapsulated Peptides, Proteins, and Other Biomolecules

“…SANS was therefore used to examine the scattering of the transmembrane peptide WALP21 encapsulated within the contrast-matched cubic phases of PE. Previous research by us has shown that the purely hydrophobic WALP21 peptide does not have much effect on the diamond cubic Q II D phase lattice parameters of PE, presumably due to minimal hydrophobic mismatch between the cylindrically shaped peptide (∼31 Å long) and the lipid bilayer thickness (∼26 Å) …”

“…We suggest that the high viscosity of the gyroid cubic phase samples, combined with the extremely low hydration (13% w/w), has precluded peptide uptake in this case. The effect is exacerbated by the high lateral pressure profile for branched chain lipids such as PE, which may also cause slow equilibration of the samples . In this case the peptide may be present as microcrystals or clusters of peptide coexisting with, but not incorporated within, the gyroid cubic phase.…”

“…The scattering intensity increased with increasing WALP21 concentration due to the additional hydrogenated material present (form factor of the peptide molecules). Corresponding SAXS curves (Figure B) showed a slight decrease in Q II D lattice parameters with 1.7 mol % WALP21, represented by a slight shift of the Bragg peaks to higher q values, in agreement with previous work …”

Direct demonstration of lipid phosphorylation in the lipid bilayer of the biomimetic bicontinuous cubic phase using the confined enzyme lipid A phosphoethanolamine transferase

“…A number of recent studies have therefore investigated changes to the lipidic nanostructure caused by the incorporation of amphiphilic proteins and peptides within the bicontinuous cubic phases. 1,[12][13][14][15][16][17] Hydrophobic mismatch, hydrophilic domain size and charge were found to be the most important factors affecting the nanostructure.…”

“…While spectroscopic techniques that investigate the conformation of the proteins have been used as an indirect measure for protein activity, 13,15,18,19 direct measurements of the activity of amphiphilic proteins within the bicontinuous cubic phase remain challenging. The enzyme activity of soluble oxidases in the nanoscale water channels of the cubic phase has been reported, for example for glucose oxidase, 10,20,21 ceruloplasmin, 20 lactate oxidase, 21 urease, 21 creatinine deiminase, 21 cholesterol oxidase, 10 horseradish peroxidase 22,23 and laccase.…”