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 extracellular media. This issue involves the basic transmembrane topology as well as more subtle variations in surface accessibility. Direct methods to evaluate the degree of exposure for residues in functional proteins expressed in living cells would be highly valuable. We sought to develop a new experimental method to determine highly surface-exposed residues, and thus transmembrane topology of membrane proteins expressed in Xenopus oocytes.


RESULTS
We have used the in vivo nonsense suppression technique to incorporate biotinylated unnatural amino acids into functional ion channels expressed in Xenopus oocytes. Binding of 125I-streptavidin to biotinylated receptors was used to determine the surface exposure of individual amino acids. In particular, we studied the main immunogenic region of the nicotinic acetylcholine receptor. The biotin-containing amino acid biocytin was efficiently incorporated into five sites in the main immunogenic region and extracellular streptavidin bound to one residue in particular, alpha 70. The position of alpha 70 as highly exposed on the receptor surface was thus established.


CONCLUSIONS
The in vivo nonsense suppression technique has been extended 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.
Gallivan
J P
JP
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.
Lester
H A
HA
Dougherty
D A
DA
D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D006031:Glycosylation; D016056:Immunodominant Epitopes / Q000378:metabolism; D007457:Iodine Radioisotopes; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D011978:Receptors, Nicotinic / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014982:Xenopus laevis
D000596:Amino Acids; D016056:Immunodominant Epitopes; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D011978:Receptors, Nicotinic; D014443:Tyrosine; C013411:biocytin; D008239:Lysine; D003545:Cysteine