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Understanding the role of backbone-backbone hydrogen bonding in the folding and stability of the hexameric enzyme 4-oxalocrotonate tautomerase (4OT)

Record Type:	Electronic resources : Monograph/item
Title/Author:	Understanding the role of backbone-backbone hydrogen bonding in the folding and stability of the hexameric enzyme 4-oxalocrotonate tautomerase (4OT)
Author:	Silinski, Peter.
Description:	208 p.
Notes:	Source: Dissertation Abstracts International, Volume: 64-11, Section: B, page: 5498.
Notes:	Supervisor: Michael C. Fitzgerald.
Contained By:	Dissertation Abstracts International64-11B.
Subject:	Chemistry, Analytical.
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3111196
ISBN:	0496587269

Understanding the role of backbone-backbone hydrogen bonding in the folding and stability of the hexameric enzyme 4-oxalocrotonate tautomerase (4OT)
Silinski, Peter.

Understanding the role of backbone-backbone hydrogen bonding in the folding and stability of the hexameric enzyme 4-oxalocrotonate tautomerase (4OT) [electronic resource] - 208 p.

Source: Dissertation Abstracts International, Volume: 64-11, Section: B, page: 5498.

Thesis (Ph.D.)--Duke University, 2003.

Backbone-backbone hydrogen bonds are ubiquitous in native protein structures. The contribution of these non-covalent interactions to protein folding and stability is not well understood. This work describes the total chemical synthesis of a number of analogues of 4-oxalocrotonate tautomerase (4OT) that contain backbone amide-to-ester mutations at specific positions in the protein's polypeptide chain. These analogues were designed to delete specific backbone-backbone hydrogen bonding interactions from the native 4OT hexamer. The biophysical properties of these analogues were assayed and compared in order to address the importance of backbone-backbone hydrogen bonding in the folding, association, stability, and function of 4OT.

ISBN: 0496587269Subjects--Topical Terms:

224793
Chemistry, Analytical.

Understanding the role of backbone-backbone hydrogen bonding in the folding and stability of the hexameric enzyme 4-oxalocrotonate tautomerase (4OT)
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245 1 0 $a Understanding the role of backbone-backbone hydrogen bonding in the folding and stability of the hexameric enzyme 4-oxalocrotonate tautomerase (4OT) $h [electronic resource]
300 $a 208 p.
500 $a Source: Dissertation Abstracts International, Volume: 64-11, Section: B, page: 5498.
500 $a Supervisor: Michael C. Fitzgerald.
502 $a Thesis (Ph.D.)--Duke University, 2003.
520 # $a Backbone-backbone hydrogen bonds are ubiquitous in native protein structures. The contribution of these non-covalent interactions to protein folding and stability is not well understood. This work describes the total chemical synthesis of a number of analogues of 4-oxalocrotonate tautomerase (4OT) that contain backbone amide-to-ester mutations at specific positions in the protein's polypeptide chain. These analogues were designed to delete specific backbone-backbone hydrogen bonding interactions from the native 4OT hexamer. The biophysical properties of these analogues were assayed and compared in order to address the importance of backbone-backbone hydrogen bonding in the folding, association, stability, and function of 4OT.
520 # $a Described here is the first detailed biophysical characterization of the folding and thermodynamic stability of 4OT, which is a new model system for protein folding studies. GuHCl-induced equilibrium unfolding data obtained on 4OT at pH 8.5 is well modeled by a two-state unfolding process involving folded hexamer and unfolded monomer. At pH 8.5, the hexamer is stabilized by 68.3 +/- 3.2 kcal · mol-1 relative to unfolded monomer. In contrast, GuHCl-induced equilibrium unfolding data obtained at lower pH are consistent with a multi-state unfolding process involving a catalytically inactive, partially folded intermediate. This intermediate can be isolated at pH 4.8. GuHCl-induced equilibrium unfolding experiments at this pH confirmed that the intermediate state is a highly structured 4OT dimer that is stabilized by 11.7 +/- 0.1 kcal · mol-1 .
520 # $a Sixteen backbone amide-to-ester mutations were systematically incorporated into the polypeptide chain of 4OT in order to delete specific backbone-backbone hydrogen bonds from the alpha-helix and beta-sheet regions of the native hexamer. Comparative analyses of the properties of these analogues revealed the following: (1) amide-to-ester mutations located in similar positions and chemical environments in the beta-sheet of 4OT destabilized the hexamer to the same degree; (2) amide-to-ester mutations near the center of the alpha-helix and beta-sheet destabilized the hexamer more than mutations near the ends of the alpha-helix and beta-sheet; and (3) amide-to-ester mutations at certain positions affect the relative populations of species that are present at equilibrium. The thermodynamic effects of these amide-to-ester mutations help to define the contribution of backbone-backbone hydrogen bonds to protein folding reactions.
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650 # 0 $a Biophysics, General. $3 226901
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710 0 # $a Duke University. $3 226880
773 0 # $g 64-11B. $t Dissertation Abstracts International
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790 1 0 $a Fitzgerald, Michael C., $e advisor
791 $a Ph.D.
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