Molecular characterization of conformational intermediates in the unfolding and dissociation of bovine plasma amine oxidase.
Item
-
Title
-
Molecular characterization of conformational intermediates in the unfolding and dissociation of bovine plasma amine oxidase.
-
Identifier
-
AAI3187450
-
identifier
-
3187450
-
Creator
-
Ramsook, Caleen B.
-
Contributor
-
Adviser: Lesley Davenport
-
Date
-
2005
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Chemistry, Biochemistry
-
Abstract
-
Improperly folded proteins have been implicated in several diseases and an understanding of the pathways that lead to the formation of the misfolded conformation may lead to a better understanding of protein folding diseases. The mechanism of protein folding for a multidomain dimer is complex and the interactions that stabilize dimeric proteins are derived from intersubunit and intrasubunit interactions. The focus of this study is to monitor the unfolding and dissociation of homodimeric bovine plasma amine oxidase (BPAO) using the chemical denaturants: guanidine hydrochloride (GuHCl), urea and sodium dodecyl sulfate (SDS).;BPAO has a molecular weight of about 192kD. Each subunit contains a copper and 2, 4, 5-trihydroxyphenylalanine quinone (TPQ) cofactors at the active site that aid in the oxidative deamination of biogenic amines to the corresponding aldehydes. The x-ray crystal structure shows that each active site is composed of residues from both monomers and there are two arms that extend from one monomer to the other that aid in the intermolecular interaction.;Each denaturant interacts with BPAO differently and therefore, the pathways for denaturation are different. Spectroscopic, chromatographic and electrophoretic techniques were used to study the denaturants interactions with BPAO and different pathways of denaturation have been proposed. The addition of SDS leads to the dissociation of the homodimer and then unfolding of the monomers, with a 12.7Kcal/mol free energy of denaturation. Urea interacts with the dimer by unfolding it into several dimeric intermediates that are not molten globule in character, and at high concentrations, it promotes dissociation. The free energy of denaturation calculated is 4.75Kcal/mol. GuHCl interacts with BPAO by the formation of a molten globule dimer, which then undergoes dissociation to form a molten globule monomeric intermediate. The free energy of denaturation in GuHCl is 15.3Kcal/mol, which is similar to that in SDS but much higher than in urea. Therefore, final conformation of the protein in GuHCl and SDS are similarly denatured, while BPAO in urea is only partially unfolded.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
