Expanding the metallomics toolbox: Development of chemical and biological methods in understanding copper biochemistry

dc.contributor.advisor Fahrni, Christoph J.
dc.contributor.author Bagchi, Pritha
dc.contributor.committeeMember Lieberman, Raquel
dc.contributor.committeeMember Kelly, Wendy L.
dc.contributor.committeeMember Williams, Loren D.
dc.contributor.committeeMember Kemp, Melissa L.
dc.contributor.department Chemistry and Biochemistry
dc.date.accessioned 2014-08-27T13:31:41Z
dc.date.available 2014-08-28T05:30:04Z
dc.date.created 2013-08
dc.date.issued 2013-05-17
dc.date.submitted August 2013
dc.date.updated 2014-08-27T13:31:41Z
dc.description.abstract Copper is an essential trace element and required for various biological processes, but free copper is toxic. Therefore, copper is tightly regulated in living cells and disruptions in this homeostatic machinery are implicated in numerous diseases. The current understanding of copper homeostasis is substantial but incomplete, particularly in regard to storage and exchange at the subcellular level. Intracellular copper is primarily present in the monovalent oxidation state. Therefore, copper(I) selective fluorescent probes can be utilized for imaging exchangeable copper ions in live cells, but these probes are often lipophilic and hence poorly water soluble. To address this problem, water-soluble fluorescent probes with greatly improved contrast ratio and fluorescence quantum yield are characterized in this work. This work also describes a novel application of water-soluble fluorescent probes, in-gel detection of copper proteins with solvent accessible Cu(I) sites under non-denaturing conditions. Knowledge of copper(I) stability constants of proteins is important to elucidate the mechanisms of cellular copper homeostasis. Due to the high affinity of most Cu(I)-binding proteins, the stability constants cannot be determined directly by titration of the apo-protein with Cu(I). Therefore, accurate determination of Cu(I) stability constants of proteins critically depends on the Cu(I) affinity standards. However, the previously reported binding affinity values of the frequently used Cu(I) affinity standards are largely inconsistent impeding reliable data acquisition for the Cu(I) stability constants of proteins. To solve this problem, a set of water-soluble ligands are developed in this work that form colorless, air-stable copper(I)-complexes with 1:1 stoichiometry. These ligands can be applied as copper(I) buffering agents and affinity standards in order to study copper biochemistry. Copper(I) binding proteins are an integral part of the copper homeostatic machinery and they work in conjunction to regulate copper uptake, distribution, and excretion. However, available evidence indicates the existence of putative copper-binding proteins that are yet to be characterized. Therefore, several proteomics-based methods are developed in this work by employing the strategy to label Cu(I)-binding cysteines in a copper-dependent manner which lays the foundation for the identification of new copper proteins from cellular extracts.
dc.description.degree Ph.D.
dc.embargo.terms 2014-08-01
dc.format.mimetype application/pdf
dc.identifier.uri http://hdl.handle.net/1853/52160
dc.language.iso en_US
dc.publisher Georgia Institute of Technology
dc.subject Copper(I) affinity standards
dc.subject Copper(I) fluorescent probes
dc.subject Differential labeling of cysteines
dc.title Expanding the metallomics toolbox: Development of chemical and biological methods in understanding copper biochemistry
dc.type Text
dc.type.genre Dissertation
dspace.entity.type Publication
local.contributor.advisor Fahrni, Christoph J.
local.contributor.corporatename School of Chemistry and Biochemistry
local.contributor.corporatename College of Sciences
relation.isAdvisorOfPublication 582e68ff-4689-4d69-95bd-97bbaf927034
relation.isOrgUnitOfPublication f1725b93-3ab8-4c47-a4c3-3596c03d6f1e
relation.isOrgUnitOfPublication 85042be6-2d68-4e07-b384-e1f908fae48a
thesis.degree.level Doctoral
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
7.16 MB
Adobe Portable Document Format
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
3.87 KB
Plain Text