Reconstituting Membrane Proteins Into Artificial Membranes and Detection of Their Activities

Abstract

We have successfully purified BR from purple membrane of Halobacterium Salinarium and Cox from the genetically engineered plasmid inserted in Rhodobacter Sphaeroides. The activities of the purified enzymes have shown in lipid vesicles as well as in polymer vesicles and planar membranes. Phosphatidylcholine derived lipid vesicles created the most nature like environment for the enzymes. Triblock copolymer membrane was the alternative choice for membrane protein reconstitution since polymers are more durable, ideal for industrial applications and support enzyme activities better. We also demonstrated the backward function of Cox in vitro by changing proton concentration in the surrounding medium. Langmuir-Blodgett method was used to reconstitute the enzymes into the planar lipid or polymer membranes. The enzyme activities of the enzymes in planar membrane system were tested by impedance spectroscopy.

Fabrication of biomolecule–copolymer hybrid nanovesicles as energy conversion systems

“…Furthermore, upon the incubation of BR-functionalized proteopolymersomes with green light in low surrounding pH conditions (3.0), it was observed that the pH level within the polymeric vesicle increased, indicating a net outward flow of protons due to an achieved 'physiological' orientation of the protein (figure 5). This pH-induced orientation of BR has been previously shown to be due to a modified surface polarity of the membrane that favours the physiological orientation of the protein [19,39]. This orientation is noted in the negative pH change with respects to the internal pH of the vesicle, signalling an outward flow of protons.…”

“…This culture broth was spun down in a Beckman JLA 9.1 rotor at 8000 rpm for 30 min to produce cell pellets. Pellets were re-suspended in 4.2 mM MgSO 4 solution with 0.1 mg ml −1 DNase I and stirred for 24 h at A depiction of the vesicle assembly is shown here as described in [6,19]. Bacteriorhodopsin (BR) is the element that confers light-dependence upon the system.…”

“…It was also observed that pH changes could be increased by crosslinking the polymer membrane using shortwave (254 nm) UV light (figure 5). This was believed to increase protein functionality due to increased mechanical stability of the membrane as well as decreased shielding of the BR from light [19,21]. Furthermore, upon the incubation of BR-functionalized proteopolymersomes with green light in low surrounding pH conditions (3.0), it was observed that the pH level within the polymeric vesicle increased, indicating a net outward flow of protons due to an achieved 'physiological' orientation of the protein (figure 5).…”

“…As such, the advantages of utilizing membrane-bound energy transducers as the core functional component of the system were further enhanced by integrating them into endgroup-stabilized ABA triblock copolymers that possess similar thicknesses to natural lipid membranes (∼4 nm, ∼5000 daltons) which allowed for transmembrane water transport and constant replenishing of the electron source for release by COX (figure 1(A)). In addition, several studies have confirmed that membrane protein functionality is retained within these membranes while allowing the proteins to withstand low pH environments [19,21]. They also possess the added advantages of customizable block compositions and block lengths based upon desired applications.…”

“…Furthermore, given that the protein-coupling process is gradient dependent, a vesicular configuration allowed for a more robust system not subject to potential system disruption due to defects commonly observed in planar systems. Following polymeric characterization, the functional protein components were integrated according to the BRinduced unidirectional transport of protons to the vesicle exterior, and accomplished through a pH gradient-dependent vectorial insertion modality [19]. Vectorial catalysis by BR was then utilized to drive COX in a partially reversed reaction, altering its normal proton pumping mechanism, to become a source of electron release.…”