Formation and characteristics of mixed lipid/polymer membranes on a crystalline surface-layer protein lattice

Abstract

The implementation of self-assembled biomolecules on solid materials, in particular, sensor and electrode surfaces, gains increasing importance for the design of stable functional platforms, bioinspired materials, and biosensors. The present study reports on the formation of a planar hybrid lipid/polymer membrane on a crystalline surface layer protein (SLP) lattice. The latter acts as a connecting layer linking the biomolecules to the inorganic base plate. In this approach, chemically bound lipids provided hydrophobic anchoring moieties for the hybrid lipid/polymer membrane on the recrystallized SLP lattice. The rapid solvent exchange technique was the method of choice to generate the planar hybrid lipid/polymer membrane on the SLP lattice. The formation process and completeness of the latter were investigated by quartz crystal microbalance with dissipation monitoring and by an enzymatic assay using the protease subtilisin A, respectively. The present data provide evidence for the formation of a hybrid lipid/polymer membrane on an S-layer lattice with a diblock copolymer content of 30%. The hybrid lipid/polymer showed a higher stiffness compared to the pure lipid bilayer. Most interestingly, both the pure and hybrid membrane prevented the proteolytic degradation of the underlying S-layer protein by the action of subtilisin A. Hence, these results provide evidence for the formation of defect-free membranes anchored to the S-layer lattice.

Fig. 1

Schematic drawing of the formation of the mixed lipid/block copolymer layer on an S-layer lattice. (a) Gold-coated QCM-D sensor crystal onto which (b) S-layer protein was recrystallized. (c) Chemically coupled phospholipids on the S-layer protein provided hydrophobic anchoring points. The structure shown in (c) was incubated with an ethanolic solution comprising lipid and polymer. (d) Finally, this solution was rapidly exchanged by buffer to form a mixed lipid/polymer membrane on the S-layer lattice.

Fig. 2

Shift in frequency (blue) and dissipation (red) during the formation process of the S-layer (SbpA) lattice and the subsequent treatment with subtilisin A (at time = 176 min).

Fig. 3

Shift in frequency (blue) and dissipation (red) during the formation of the S-layer supported lipid membrane and the subsequent treatment with subtilisin A. (A) Injection of the SbpA solution; (B) activation of SbpA; (C) addition of anchor lipids (DMPE); (D) rinsing with 70% ethanol; (E) addition of membraneforming lipids (DOPC) in ethanol; (F) rapid solvent exchange from organic to aqueous phase to form a membrane; (G) addition of subtilisin A; and (H) rinsing with HEPES buffer.

Fig. 4

Shift in frequency (blue) and dissipation (red) during the formation process of the S-layer supported mixed lipid/polymer membrane and the subsequent treatment with subtilisin A. (A) Injection of the SbpA solution; (B) activation of SbpA; (C) addition of anchor lipids (DMPE); (D) rinsing with 70% ethanol; (E) addition of lipid/polymer mixture (DOPC/PBD-PEO) in ethanol; (F) rapid solvent exchange from organic to aqueous phase to form a hybrid membrane; (G) addition of subtilisin A; and (H) rinsing with HEPES buffer.