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Biophys. J. BioFAST: First Published December 30, 2004. doi:10.1529/biophysj.104.052944
© 2004 by the Biophysical Society.

A more recent version of this article appeared on March 1, 2005.
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CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING

Role of Glycosylation and Membrane Environment in Nicotinic Acetylcholine Receptor Stability

Corrie J. B. daCosta 1, Daniel E. E. Kaiser 1 and John E. Baenziger 1*

1 University of Ottawa

* To whom correspondence should be addressed. E-mail: jebaenz{at}uottawa.ca.

Submitted on September 21, 2004
Revised on November 12, 2004
Accepted on 20 December 2004


  Abstract
The effects of glycosylation and membrane environment on the structural stability of the nicotinic acetylcholine receptor (nAChR) from Torpedo have been investigated in order to improve our understanding of factors that influence eukaryotic membrane protein crystallization. Gel shift assays and carbohydrate specific staining show that the deglycosylation enzyme, Endo F1, removes at least 50% of membrane reconstituted nAChR glycosylation. The extent of deglycosylation with Endo F1 increases upon detergent solubilization. Removal of between 60-100% of high mannose moieties from the nAChR has no effect on nAChR secondary structure, stability, or flexibility. Deglycosylation does not influence either agonist binding or the ability of the nAChR to undergo agonist induced conformational change. In contrast, nAChR structural stability, flexibility, and function are all negatively influenced by simple changes in reconstituted membrane lipid composition. Our results suggest that deglycosylation may represent a feasible approach for enhancing the crystallizability of the nAChR. Our data also demonstrate that the dependence of nAChR structural stability on lipid environment may represent a significant obstacle to nAChR crystallization. Some membrane proteins may have evolved complex interactions with their lipid environments. Understanding the complexity of these interactions may be essential for devising an appropriate strategy for the crystallization of some membrane proteins.

Key Words: FTIR Spectroscopy, Hydrogen-Deuterium Exchange Kintetics, Lipid-Protein Interactions, Thermal Denaturation, deglycosylation, nAChR




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Copyright © 2004 by the Biophysical Society.