A Platform for Presenting and Characterizing Membrane Proteins
- Fig. 1: The two principles of artificial cell membrane (ACM) technology: Self-assembly of a prototypical block copolymer into vesicles, and in-vitro synthesis from an optimized cell-free extract. The complementary DNA (cDNA) encoding the protein of interest is added to a polymersome suspension. The protein co-translationally inserts into the polymer membrane, which provides a native-like physical environment for proper folding.
- Fig. 2: a. In-vitro expression and integration of Cld2 into polymersomes. The Western blot images show bands corresponding to Cldn2 synthesized in the absence (1) and presence of polymersomes (3), as well as in the presence of liposomes (2). b. Schematic representation of the SPR binding experiment. c. The Cldn2 polymersomes show superior binding as compared to liposomes.
- Fig. 3: left: Predicted structure of DRD2 in complex with dopamine (rendered as spheres), middle: Dot plot of the binding of dansyl dopamine to DRD2 in polymersome membranes. P3 indicates fluorescence from dansyl dopamine bound to the proteopolymersomes. The bar graph shows the relative fluorescence of this sample  versus that of pure polymersomes , right: Surface immobilized DRD2 polymersomes as visualized by the binding of fluorescent dansyl-dopamine. The ligand showed dosedependent replacement.
Membrane Protein Research: The past decade has seen great progress in the characterization and structural analysis of soluble proteins, with protein-ligand interactions now routinely being analyzed by spectrometry (X-ray, NMR), surface plasmon resonance (SPR), and computational methods. Although successful for soluble proteins, these methods have lagged behind in the elucidation of an essential class of proteins that are embedded in the cell membrane, i.e., membrane proteins, as exemplified by the number of membrane protein structures deposited in the protein data base, which amounts to around 600 out of 100,000 published structures. Membrane proteins are essential because they relay the chemical cues that make the respective cell an integral part of its surroundings, be it a tissue, organ, or brain. Successful targeting of membrane proteins by appropriate therapeutic strategies often translates into new medicines, which is why a considerable fraction of block-buster drugs on the market today target this class of proteins.
The reason why membrane proteins have eluded thorough characterization is their instability in the absence of a membrane. During protein translation, membrane proteins require a bilayer membrane to fold correctly. This membrane is typically formed from a lipid shell, which also forms the principal architecture of the plasma membrane. The first medium of choice for studying membrane proteins, therefore, has been whole cells designed to overexpress the membrane protein target, or isolated membrane fractions derived from these cells. The use of cell culture is time-consuming and not technically straightforward, however, as it requires a sophisticated infrastructure and high level of specialized skills to store, grow, and maintain such cell lines, even before the start of protein expression. Not all membrane proteins tend to express at their true physiological levels under these conditions, and certain classes that are toxic to the cell cannot be produced at all.
Finally, cells are notorious for exhibiting significant batch-to-batch variation, and are prone to false positives due to the heterogeneous nature of the cellular membrane, and interference by other proteins in the expression and translocation to the cell surface.
In attempts to circumvent the use of cells, techniques have been developed to demonstrate that membrane proteins, free from interfering elements, may be reconstituted, or even directly expressed into pure membranes (i.e., liposomes). This strategy still does not improve the stability of the protein since lipid membranes themselves are labile assemblies, at least for realistic technological applications. Artificial cell membrane (ACM) technology presents a two-sided solution to the problems associated with membrane proteins. First, it allows for rapid production (within 1-2 days) of high quantities of membrane proteins in a format that bypasses the limitations imposed by whole cells. Secondly, the expressed protein is embedded into a stable, monodisperse polymer matrix that closely mimics the amphiphilic nature of the cellular membrane (Fig. 1). The basic component of the polymer matrix is formed from amphiphilic block copolymers that self-assemble into polymersomes, featuring a bilayer membrane analogous to lipids, yet with a much increased physical stability. On the other hand, the protein itself is produced by cell-free, in-vitro synthesis (IVS): expressing the protein using the isolated protein machinery of a cell. As a result, during translation, the protein is integrated into the block copolymer matrix, circumventing both the use of cells and the labile lipid membrane.
Probing the Expression of Membrane Proteins
One of the main areas for application of this technology is in the initial stage of drug development. In order to demonstrate the applicability, the possibility of integrating membrane proteins into block copolymer membranes was first demonstrated. As a proof-of-concept, claudin2 (Cldn-2), a comparatively simple membrane protein, was chosen. Cldn-2 is a protein involved in cell-cell interactions containing four trans-membrane helices. Expression of the protein using a wheat germ extract in the presence of polymersomes showed that Cldn-2 was readily integrated into the polymersome membrane (Fig. 2). The functionality of the embedded protein was tested by monitoring binding to a Cldn2-specific antibody using SPR, showing strong binding of polymersomes expressing Cldn2. Importantly, binding of liposomes was much reduced (due to low stability) and binding of empty polymersomes (lacking Cldn2) was absent.
Probing the Functionality of the Produced Membrane Proteins
Once we demonstrated that direct expression of membrane proteins into polymersome carriers is feasible, we focused on the in-vitro synthesis (IVS) of a class of membrane proteins known as G protein-coupled receptors (GPCRs), which constitute targets for the majority of the top blockbuster drugs on the market today. GPCRs are directly involved in numerous signal processes involved in a number of diseases ranging from cancer and HIV to obesity and depression. As a proof-of-principle, we selected the dopamine receptor 2 (DRD2) because it is a well-characterized pharmacological target that is involved in depression and Parkinson's disease. IVS of this protein in the presence of polymersomes showed the successful incorporation of the translated product. The purified polymersomes were used to assess the functionality of the membranes by a variety of ligand-binding experiments.
We first used flow cytometry to study the binding of a fluorescently labeled native ligand of DRD2 receptor, dansyl-dopamine. Flow cytometry detects the polymersomes, but not the free ligand. Incubation of polymersomes carrying the DRD2 receptor showed a significant shift in fluorescence intensity as compared to polymersomes in absence of the receptor, indicating that integrated GPCRs were able to bind polymer-embedded receptor (Fig. 3). A definite proof of the presence of correctly folded protein was demonstrated by displacement of the ligand by dopamine itself. To explore the versatility of the platform for assay development, we immobilized the membranes on a surface for the DRD2 experiments. Using such an approach with lipids would involve cumbersome surface functionalization and conjugation methodologies for liposomes so as to maintain the fragile bilayer. The artificial membranes, however, were readily immobilized using a stamping procedure, demonstrating their physical stability. After immobilization the fluorescent ligand bound to the receptor was subjected to a displacement assay, by incubating with increasing concentrations of (unlabeled) dopamine. After a rinsing step the fluorescence intensity of the surfaces was measured, showing a clear decrease in fluorescence intensity as a function of unlabeled dopamine concentration, indicating effective binding of dopamine on the receptor.
We are currently investigating the performance of D2 receptors produced in ACMs in the screening of a library of small-molecule compounds, as compared to receptor produced in native cell membranes.
Using claudin and the dopamine receptor as examples, we have shown that in-vitro expression of membrane proteins in synthetic block copolymer membranes is feasible, yielding receptors that show binding of native ligands. The platform can readily be optimized for applications such as SPR and array-based screening. Importantly, the in-vitro approach offers the possibility of expressing any membrane protein of which the cDNA is available, within a time frame of a few days. We are currently integrating ACM technology with various high-throughput assay methods based on radioligand-binding, fluorescence and label-free techniques.
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