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Chemical-scale manipulation of ion channels: in vivo nonsense suppression and targeted disulfide crosslinking
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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.
Authors :
Zacharias, Niki Marie
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David Tirrell, Jackie Barton, Richard Roberts, Henry Lester, Dennis Dougherty
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Publisher :  California Institute of Technology     Type :  text     Format :  application/pdf    
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restricted, I hereby certify that, if appropriate, I have obtained a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to California Institute of Technology or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.
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