Tyler Collins

Defense Date

10-9-2009

Graduation Date

2009

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

Committee Chair

Charles Dameron

Committee Member

Jeffry Madura

Committee Member

Mihaela Mihailescu

Committee Member

Jana Patton-Vogt

Keywords

CopY, Dimerization, ITC, Zinc

Abstract

Dimerization of cellular proteins has become an intense field of study due to its importance in signal transduction and gene expression, amongst many other functions. Arguably the most intriguing quality regarding dimerization is the high incidence of simultaneous dimeric events occurring within the cell, where each constructed dimer individually controls a single pathway with high specificity. Additionally, the discoveries of new motifs possessing this seemingly common functionality participate in truly unique and often poorly understood mechanisms. CopY, a metal-responsive bacterial repressor from Enterococcus hirae possesses distinctive dimerization properties. We show that this cysteine metal-binding motif, which contains a metal-reactive C-terminal -CxCxxxxCxC- site used for sensing high Cu(I), is also indispensible for dimer formation in its Zn(II) form in vivo and in vitro. Paired adjacent to the cysteine motif is a repeating aliphatic sequence that mimics a coiled-coil, where the two combined contributors produce a dimer of low micromolar affinity. Removal of the metal-binding site perturbs the dimer's stability, resulting in the loss of specific two-stranded dimerization and shifting the complexes equilibrium towards an aggregated state according to isothermal titration calorimetry. This transition illuminates the site's role in organizing quaternary structure. Protein-fragment complementation assay technology was employed to explore the whole dimer's ability to heterodimerize in an effort to establish the level of specificity within the aliphatic-repeat sequence. The results indicate that symmetrical deletions within the aliphatic-repeat produce stronger dimers compared to those asymmetrically constructed. In all cases, metal-binding site was necessary for successful construction of high-affinity dimers according to both methods.

Format

PDF

Language

English

Download

Share

COinS