A Novel Method to Probe Membrane Protein Topology Using Unnatural Amino Acid Mutagenesis and Antibody Epitope Tagging

Abstract

We have developed a novel strategy to probe the topology of membrane proteins in their native bilayer environment. Our technique relies on unnatural amino acid mutagenesis to incorporate p-azido-L-phenlyalanine at a specific site in the expressed target protein. The reactive azido moiety facilitates Staudinger-Bertozzi ligation chemistry to introduce a monoclonal antibody (mAb) epitope-tagged phosphine derivative. This site-specific labeling method allows the flexibility and precision of single codon scanning, and appears to be superior to current biochemical approaches that rely on chemical modifications and/or introduction of epitope tags by mutagenesis. Our approach can be used to identify gross topological determinants for transmembrane proteins of unknown topology as well as to elucidate secondary structural elements with chemical precision. We demonstrate the experimental feasibility of our technique on human C-C chemokine receptor 5 (CCR5), a heptahelical transmembrane G protein-coupled receptor (GPCR) of known topology. CCR5 is a major co-receptor for HIV-1 entry into host cells. We labeled CCR5 in membranes with FLAG peptide epitope-phosphine at various sites on the receptor's intra- and extracellular surfaces. The differential reactivity of the FLAG epitope-phosphine reagent for the azido group on the various CCR5 mutants correlated to the known topology of CCR5 and defined specific helical boundaries. We further applied the new label/probe technology to mammalian cells in culture in order to label extracellular sites on surface-expressed CCR5. Our new method appears to be satisfactory to probe membrane protein topology in polytopic membrane proteins and has potential applications in the study of receptor signaling mechanisms in live cells.