Solubilizing an Integral Membrane Protein using Site-directed Mutagenesis

There have been only a few successes in determining the structure of
membrane proteins in spite of the hundreds of membrane proteins awaiting a structural solution. The major bottleneck arises from the difficulty in producing crystals that diffract to a high resolution, and this is a result of the slippery lipophilic regions of the proteins that do not readily form solid crystal contacts. One possible route around this problem is to place some charged amino acids in these regions to solubilize the protein away from the membrane, making the protein both easier to purify and easier to crystallize. We are exploring this possibility using a group of membrane proteins that bind to only one leaflet of the lipid bilayer, the so-called interfacial integral membrane proteins. After identifying the membrane-binding domains using deletion mutagenesis, these domains can be targeted by site-directed mutagenesis (with input from homology modeling) to release the protein from the membrane.This project will provide an introduction to the standard methods of molecular biology, as well as give experience in protein expression, cell fractionation, and protein analysis using polyacrylamide gels and western immunoblotting.