Structural and Biochemical Studies of the Metal Binding Protein CusF and its Role in Escherichia coli Copper Homeostasis

Persistent Link:
http://hdl.handle.net/10150/193875
Title:
Structural and Biochemical Studies of the Metal Binding Protein CusF and its Role in Escherichia coli Copper Homeostasis
Author:
Loftin, Isabell
Issue Date:
2008
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Biometals such as copper, cobalt and zinc are essential to life. These transition metals are used as cofactors in many enzymes. Nonetheless, these metals cause deleterious effects if their intracellular concentration exceeds the cells' requirement. Prokaryotic organisms usually employ efflux systems to maintain metals in appropriate intracellular concentrations.The Cus system of Escherichia coli plays a crucial part in the copper homeostasis of the organism. This system is a tetrapartite efflux system, which includes an additional component compared to similar efflux systems. This fourth component is a small periplasmic protein, CusF. CusF is essential for full copper resistance, yet its role within the Cus system has not been characterized. It could potentially serve in the role of a metallochaperone or as a regulator to the Cus system.To gain insight into the molecular mechanism of resistance of this system, I have structurally and biochemically characterized CusF. Using X-ray crystallography I determined the CusF structure. CusF displays a novel fold for a copper binding protein. Through multiple sequence alignment and NMR chemical shift experiments, I proposed a metal binding site in CusF, which I confirmed through determination of the structure of CusF-Ag(I). CusF displays a novel coordination of Ag(I) and Cu(I) through a Met2His motif and a cation-pi interaction between the metal ion and a tryptophan sidechain. Furthermore, I have shown that CusF binds Cu(I) and Ag(I) specifically and tightly.I investigated the role of the tryptophan at the binding site to establish its effect on metal binding and function of CusF. I have shown through competitive binding assays, NMR studies and through collaborative EXAFS studies that the tryptophan plays an essential role in CusF metal handling. The affinity of CusF for Cu(I) is influenced by this residue. Moreover, the tryptophan also caps the binding site such that oxidation of the bound metal as well access to adventitious ligands is prevented. In summary, these findings show that the structure and metal site of CusF are unique and are specifically designed to perform the function of CusF as a metallochaperone to the Cus system.
Type:
text; Electronic Dissertation
Keywords:
copper; metallochaperone; periplasmic protein; copper chaperone; copper homeostasis; protein structure
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Biochemistry & Molecular Biophysics; Graduate College
Degree Grantor:
University of Arizona
Advisor:
McEvoy, Megan M.; Montfort, William R.
Committee Chair:
McEvoy, Megan M.; Montfort, William R.

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleStructural and Biochemical Studies of the Metal Binding Protein CusF and its Role in Escherichia coli Copper Homeostasisen_US
dc.creatorLoftin, Isabellen_US
dc.contributor.authorLoftin, Isabellen_US
dc.date.issued2008en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractBiometals such as copper, cobalt and zinc are essential to life. These transition metals are used as cofactors in many enzymes. Nonetheless, these metals cause deleterious effects if their intracellular concentration exceeds the cells' requirement. Prokaryotic organisms usually employ efflux systems to maintain metals in appropriate intracellular concentrations.The Cus system of Escherichia coli plays a crucial part in the copper homeostasis of the organism. This system is a tetrapartite efflux system, which includes an additional component compared to similar efflux systems. This fourth component is a small periplasmic protein, CusF. CusF is essential for full copper resistance, yet its role within the Cus system has not been characterized. It could potentially serve in the role of a metallochaperone or as a regulator to the Cus system.To gain insight into the molecular mechanism of resistance of this system, I have structurally and biochemically characterized CusF. Using X-ray crystallography I determined the CusF structure. CusF displays a novel fold for a copper binding protein. Through multiple sequence alignment and NMR chemical shift experiments, I proposed a metal binding site in CusF, which I confirmed through determination of the structure of CusF-Ag(I). CusF displays a novel coordination of Ag(I) and Cu(I) through a Met2His motif and a cation-pi interaction between the metal ion and a tryptophan sidechain. Furthermore, I have shown that CusF binds Cu(I) and Ag(I) specifically and tightly.I investigated the role of the tryptophan at the binding site to establish its effect on metal binding and function of CusF. I have shown through competitive binding assays, NMR studies and through collaborative EXAFS studies that the tryptophan plays an essential role in CusF metal handling. The affinity of CusF for Cu(I) is influenced by this residue. Moreover, the tryptophan also caps the binding site such that oxidation of the bound metal as well access to adventitious ligands is prevented. In summary, these findings show that the structure and metal site of CusF are unique and are specifically designed to perform the function of CusF as a metallochaperone to the Cus system.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectcopperen_US
dc.subjectmetallochaperoneen_US
dc.subjectperiplasmic proteinen_US
dc.subjectcopper chaperoneen_US
dc.subjectcopper homeostasisen_US
dc.subjectprotein structureen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineBiochemistry & Molecular Biophysicsen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorMcEvoy, Megan M.en_US
dc.contributor.advisorMontfort, William R.en_US
dc.contributor.chairMcEvoy, Megan M.en_US
dc.contributor.chairMontfort, William R.en_US
dc.contributor.committeememberCordes, Matthew H. J.en_US
dc.contributor.committeememberHorton, Nancy C.en_US
dc.contributor.committeememberRensing, Christopheren_US
dc.contributor.committeememberWalker, F. Annen_US
dc.identifier.proquest2924en_US
dc.identifier.oclc659749948en_US
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