Membrane Insertion of Small Proteins

Proteins that are less than 10 kDa in size are easily purified under denaturing conditions and can often be refolded by removal of the denaturing agents. The purified small membrane proteins are competent for membrane insertion when the denaturing agent is diluted out and a membranous system like liposomes or proteoliposomes is added. This system allows the characterization of the membrane insertion process at the molecular level. The insertion of the protein into proteoliposomes can be followed by protease digestion and Western blot analysis. Only if the antigenic region of the protein has translocated into the lumen of the proteoliposome it is protected from the protease. When combining this approach with fluorophores that are placed within the membrane protein, membrane insertion can also be followed by fluorescence correlation spectroscopy.