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Chemical-scale manipulation of ion channels: in vivo nonsense suppression and targeted disulfide crosslinking

Citation

Zacharias, Niki Marie (2004) Chemical-scale manipulation of ion channels: in vivo nonsense suppression and targeted disulfide crosslinking. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-01042004-210542

Abstract

The study of the three-dimensional shape and structure-function relationships of ion channels is a very challenging field of research. Ion channels are integral-membrane proteins that when open allow ions to flux across the cell membrane. The structure and function of ion channels are dependent on the cell membrane that surrounds them. Because an ion channel must be embedded in a cell membrane, many techniques used to probe the structure of soluble proteins cannot be used in the study of ion channels.

One versatile technique that has been shown to be quite valuable in the structure-function studies of ion channels is the in vivo nonsense suppression method for unnatural amino acid incorporation. This technique allows one to site-specifically incorporate an unnatural amino acid or hydroxy acid into a protein in a living cell. To date more than 60 amino acids and hydroxy acids have been incorporated into proteins using in vivo nonsense suppression. The method has been shown to accommodate a wide variety of unnatural amino acids and hydroxy acids. Chapter One will discusses the in vivo nonsense suppression method in greater detail.

A key component of this work is the design and synthesis of new unnatural amino acids that have novel properties. Chapter 2 discusses the synthesis and uses of 5-(o-nitrobenzyl)selenyl-2-hydroxypentanoic acid (NBSeOH). NBSeOH is used to site-specifically cleave a peptide backbone. The o-nitrobenzyl protecting group is photochemically removed to reveal a selenium anion. The selenium anion then initiates an intramolecular SN2 displacement that cleaves the backbone of the protein. Preliminary data reveals that NBSeOH can be incorporated into a protein in vivo and in vitro, and photolysis of proteins and peptides containing NBSeOH does lead to protein backbone cleavage.

Chapter 4 discusses how the in vivo nonsense suppression method was used to incorporate unnatural amino acids containing a quaternary ammonium moiety to mimic the quaternary ammonium on acetylcholine. These unnatural amino acids were used to probe the nicotinic acetylcholine receptor?s binding site. These unnatural amino acids are called tethered agonists because when they were incorporated into four different positions on the nicotinic acetylcholine receptor partial opening of the channel occurred even when agonist was not present. These tethered agonists were used to obtain distance information about where acetylcholine binds within the receptor.

Another technique used to probe the structure of ion channels is targeted disulfide crosslinking. In the targeted disulfide crosslinking method, cysteine residues are introduced at various locations throughout a protein and oxidized to see whether disulfide bond formation can occur. Since only cysteine residues close in space will form a disulfide bond, this method can reveal fine structural aspects of a protein. The method was used to study the pore lining structure of the nicotinic acetylcholine receptor. Several cysteine mutants were made using mutagenesis and then studied in functional channels expressed in Xenopus oocytes. The channels were then exposed to oxidizing agents, and the ability of these mutant channels to form disulfide bonds was evaluated. Chapter 3 describes the work dealing with the targeted disulfide crosslinking experiments in the nicotinic acetylcholine receptor.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:in vivo nonsense suppression; ion channels; targeted disulfide crosslinking; unnatural amino acids
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Dougherty, Dennis A. (advisor)
  • Lester, Henry A. (co-advisor)
Thesis Committee:
  • Tirrell, David A. (chair)
  • Dougherty, Dennis A.
  • Lester, Henry A.
  • Barton, Jacqueline K.
  • Roberts, Richard W.
Defense Date:26 September 2003
Record Number:CaltechETD:etd-01042004-210542
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-01042004-210542
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:20
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:05 Jan 2004
Last Modified:26 Dec 2012 02:26

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