SITE-SPECIFIC INCORPORATION OF BIOTINYLATED AMINO-ACIDS TO IDENTIFY SURFACE-EXPOSED RESIDUES IN INTEGRAL MEMBRANE-PROTEINS

Background: A key structural issue for all integral membrane proteins is the exposure of individual residues to the intracellular or extrace llular media. This issue involves the basic transmembrane topology as well as more subtle variations in surface accessibility. Direct method s to evaluate the degree of exposure for residues in functional protei ns expressed in living cells would be highly valuable. We sought to de velop a new experimental method to determine highly surface-exposed re sidues, and thus transmembrane topology, of membrane proteins expresse d in Xenopus oocytes. Results: We have used the in vivo nonsense suppr ession technique to incorporate biotinylated unnatural amino acids int o functional ion channels expressed in Xenopus oocytes. Binding of I-1 25-streptavidin to biotinylated receptors was used to determine the su rface exposure of individual amino acids. In particular, we studied th e main immunogenic region of the nicotinic acetylcholine receptor. The biotin-containing amino acid biocytin was efficiently incorporated in to five sites in the main immunogenic region and extracellular strepta vidin bound to one residue in particular, alpha 70. The position of al pha 70 as highly exposed on the receptor surface was thus established. Conclusions: The in vivo nonsense suppression technique has been exte nded to provide the first in a potential series of methods to identify exposed residues and to assess their relative exposure in functional proteins expressed in Xenopus oocytes.