Purification and behavior of proteins and membrane-associated peptides through extraction via Winsor-III systems into bicontinuous microemulsions
Hayes, D. G., R. Ye, V. S. Urban, N. M. O'Neill, S. V. Pingali, V. Sharma, and R. N. Dunlap.  2018.  22nd International Symposium on Surfactants in Solutions Conference, Norman, OK, 3-8 June, 2018.  (invited)

Abstract:
Proteins can be readily isolated in the middle, bicontinuous microemulsion (BmE) phase of Winsor-III systems due to an electrostatic attractive or hydrophobic interaction-based driving force imposed by surfactants. We have successfully achieved high extraction efficiency for Winsor-III –based extraction (>75%) of several different proteins using anionic surfactant-based Winsor-III systems, leading to BmE phases highly concentrated in protein (> 10 g/L for several different proteins). The proteins reside near the surfactant head groups, as supported by results from small-angle x-ray or neutron scattering (SAXS and SANS, respectively). Proteins can be back-extracted from the BmE phase by counteracting the driving force, e.g., using aqueous stripping solutions high in salinity (to promote Debye shielding of the head groups) or using a pH greater than the pI of the proteins (for anionic surfactant-based systems). BmEs serve as biomembrane-mimetic systems due to their possession of surfactant monolayers of near-zero curvature, resembling oil-swollen phospholipid bilayers. The similarity motivated us to investigate the behavior of membrane-associated peptides and proteins in BmE Winsor-III phases. BmEs possess several advantages over other biomembrane mimetic systems, including optical transparency, isotropic nature, and high amount of surface area per volume. BmEs can also serve as transdermal and perhaps oral delivery systems. We determined that melittin, a model antimicrobial peptide possessing a broad activity against many microorganisms that promote infections (including antibiotic-resistant bacteria), behaves similarly in BmEs as in vesicles: enhanced alpha-helical folding, disruption of packing arrangement of amphiphiles (observed by SANS and reflective of melittin’s solubilization within the surfactant monolayers), and strong aggregation of melittin at high concentrations. Similarly, solubilization of alpha-synuclein in BmEs promoted a major secondary structural change in the peptide, from random coil to alpha-helix formation, and enhanced aggregation of the peptide (through SANS analysis using neutron contrast matching), events observed in other biomembrane mimetic systems that may play a role in the occurrence of Parkinson’s disease. Through quasi-elastic neutron scattering, we found that membrane-associated proteins slow both the lateral and internal motions of surfactants in the BmE monolayers. From SANS and Neutron Spin-Echo results, we found that membrane-associated proteins and peptides decrease the membrane bending constant, suggesting an increase of interfacial fluidity.