Solubilization of membrane proteins with the Peptidisc
Membrane proteins represent ~60% of medical drug targets and encompass 1/5th of the human proteome; yet these proteins are vastly under-represented in structure and interaction databases. This discrepancy is due to biochemical and biophysical studies requiring proteins in a water-soluble state, while membrane proteins are naturally sequestered in a hydrophobic lipid environment. In order to render membrane proteins into a soluble state, that is amenable for their study, researchers generally use detergents to extract and purify these proteins. However, these surfactants have many undesired effects on protein structure, function and downstream analysis.
Work from our group and others has focused on developing nanotechnology-based reconstitution systems to try replacing the natural lipid bilayer while maintaining water-solubility. These membrane mimetics, either protein-based scaffolds or synthetic polymers, have been around for a decade, yet their utilities did not reach the expected potential due to the optimization and adaptation required for each membrane protein system. Recently, our laboratory developed a “one-size-fits-all” formulation known as the Peptidisc— the Peptidisc is made by multiple copies of an amphipathic peptide that spontaneously associate around transmembrane domains of proteins upon removal of detergent. The peptide number adapts spontaneously to fit the size and shape of the protein, allowing for minimal reconstitution optimization.
The end result is a membrane protein that is stable, free of detergent effects, and soluble in aqueous solution. The Peptidisc is a new tool that we hope will allow more researchers, including those who are not expert biochemists, to study membrane proteins. This will yield a better understanding of the structure and function of the cellular membrane as it interacts with the environment. Since the approach is both simple and easy to apply, more membrane proteins may now be included in high-throughput searches for potential new drugs that may help treat various medical conditions.
By making membrane proteins soluble, and retaining their native state, structural characterization becomes possible. Utilizing cryo-electron microscopy, high-resolution structural data of membrane proteins can be obtained. Our work with cryo-EM focuses on isolating and structurally characterizing novel membrane protein complexes.
Injection of membrane proteins into animals with detergent causes aggregation and loss of relevant native protein structure for antibody generation. Hence, by using the Peptidisc to stabilize proteins in their native form, we can inject soluble membrane proteins into animals. This results in antibodies that recognize the protein of interest in its native form, upon injection of the complex.
Membrane proteins in the Peptidisc allows for the native form of a protein to be presented to a phage display library. Therefore the interactions found will be more useful, as non-native forms of the protein are excluded, and the library sorting can give biologically relevant binders faster.
As membrane proteins constitute a disproportionate fraction of drug targets, the characterization of drug interactions with target protein is very important. Because the Peptidisc holds membrane proteins in its native, functional state, the binding characteristics of substrates, as well as drugs, of the target protein can be assessed. This allows for effective and reliable Kd calculations, on and off rates, binding sites, and protein stability.