Spontaneous Reconstitution of Functional Transmembrane Proteins During Bioorthogonal Phospholipid Membrane Synthesis

Abstract

Transmembrane proteins are critical for signaling, transport, and metabolism, yet their reconstitution in synthetic membranes is often challenging. Non-enzymatic and chemoselective methods to generate phospholipid membranes in situ would be powerful tools for the incorporation of membrane proteins. Herein, the spontaneous reconstitution of functional integral membrane proteins during the de novo synthesis of biomimetic phospholipid bilayers is described. The approach takes advantage of bioorthogonal coupling reactions to generate proteoliposomes from micelle-solubilized proteins. This method was successfully used to reconstitute three different transmembrane proteins into synthetic membranes. This is the first example of the use of non-enzymatic chemical synthesis of phospholipids to prepare proteoliposomes.

Keywords: membrane proteins; phospholipids; proteoliposomes; self-assembly; synthetic biology.

Figure 1

De novo synthesis of phospholipid membranes and concurrent in situ incorporation of proteins. A) Two unique bioorthogonal routes to produce synthetic analogues of POPC. Phospholipid 3a is formed by CuAAC between alkyne lysolipid 1a and alkyl azide 2a. Phospholipid 3b is formed via NCL between cysteine lysolipid 1b and thioester 2b. B) Model for spontaneous reconstitution of transmembrane proteins during non-enzymatic phospholipid membrane formation. Protein is solubilized with synthetic mimetics of lysophosphatidylcholine, acting as the detergent to form micelle-solubilized protein complexes. Addition of the reactive alkyl precursor and subsequent coupling reaction results in the spontaneous generation of the corresponding proteoliposomes.

Figure 2

Characterization of CcO activity. A) Normalized activity of CcO determined by measuring the absorption peak of cytochrome c at 550nm. (+) DDM: CcO activity as measured in n-dodecyl β-D-maltoside (DDM) [positive control]. (-) DDM: CcO activity in DDM following detergent removal using Bio-Beads® SM-2 and dialysis [negative control]. POPC: CcO activity after being reconstituted in POPC and the removal of detergent with Bio-Beads® SM-2 and dialysis. In situ CuAAC: CcO activity after spontaneously being reconstituted in the triazole-containing phospholipid membranes. As a precaution, membranes were treated with Bio-Beads® SM-2 and dialyzed. B) Relative fluorescence units (RFUs) of the pH sensitive dye (pyranine) encapsulated in CcO proteoliposomes formed in situ by the CuAAC approach. Upon adding cytochrome c there is a gain of fluorescence, as measured at 515 nm, indicating that pyranine is being deprotonated from the shuttling of protons across the membrane.

Figure 3

Spinning disk confocal fluorescence microscopy images of spontaneously reconstituted proteoliposomes using either the CuAAC (left) or NCL (right) coupling methodologies. The lipid channel shows the location of the lipid membrane staining dye, Texas Red® DHPE. The protein channel shows the location of fluorescent transmembrane proteins [Proteins modified with Alexa Fluor® 488 NHS-ester (CcO and MsbA) or EGFP (PMCA2)]. CuAAC scale bar denotes 5 μm. NCL scale bar denotes 10 μm.

Figure 4

Monitoring the activity of the selective calcium ion transport protein PMCA2 by measuring the change in conductance across a planer lipid bilayer. A) The flux of Ca²⁺ through PMCA2 was observed in a black lipid membrane (BLM) system using synthetic phospholipid 3b. Ca²⁺ ion flux across the membrane mediated by PMCA2 was measured under an applied potential of 80 mV. B) In comparison, no ion current events were observed when Ca²⁺ ions were replaced by Na⁺ ions in the recording medium. Dash line in A corresponds to the current trace in B.