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Site-specific incorporation of biotinylated amino acids to identify surface-exposed residues in integral membrane proteins

Gallivan, Justin P. and Lester, Henry A. and Dougherty, Dennis A. (1997) Site-specific incorporation of biotinylated amino acids to identify surface-exposed residues in integral membrane proteins. Chemistry and Biology, 4 (10). pp. 739-749. ISSN 1074-5521. https://resolver.caltech.edu/CaltechAUTHORS:20200515-142830762

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Abstract

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, α70. The position of α70 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/s1074-5521(97)90312-4DOIArticle
ORCID:
AuthorORCID
Lester, Henry A.0000-0002-5470-5255
Dougherty, Dennis A.0000-0003-1464-2461
Additional Information:© 1997 Published by Elsevier Ltd. Received 24 July 1997, Accepted 27 August 1997. This work was supported by the National Institutes of Health (NS-34407 and NS-11756). J.P.G. is grateful to Caltech and to the Eastman Kodak Corporation for support in the form of graduate fellowships. We thank Scott K. Silverman for providing a sample of α-NVOC-lysine fluorenylmethyl ester.
Funders:
Funding AgencyGrant Number
NIHNS-34407
NIHNS-11756
CaltechUNSPECIFIED
Eastman Kodak CorporationUNSPECIFIED
Subject Keywords:biotin/streptavidin; in vivo suppression; surface exposure; transmembrane topology; unnatural amino acids
Issue or Number:10
Record Number:CaltechAUTHORS:20200515-142830762
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200515-142830762
Official Citation:Justin P. Gallivan, Henry A. Lester, Dennis A. Dougherty, Site-specific incorporation of biotinylated amino acids to identify surface-exposed residues in integral membrane proteins, Chemistry & Biology, Volume 4, Issue 10, 1997, Pages 739-749, ISSN 1074-5521, https://doi.org/10.1016/S1074-5521(97)90312-4. (http://www.sciencedirect.com/science/article/pii/S1074552197903124)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:103246
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:15 May 2020 21:38
Last Modified:15 May 2020 21:38

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