Date of Award

12-2014

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Dr. David Huffman

Second Advisor

Dr. Ramakrishna Guda

Third Advisor

Dr. Sherine Obare

Fourth Advisor

Dr. Charles Ide

Keywords

Wilson Protein, GFP, two-photon absorption, protein unfolding, circular dichroism

Abstract

Wilson disease protein is a copper-transporting P1B type ATPase. It has large N-terminal copper binding domain which is composed of six homologous sub-domains. Each of these six domains is ~72 residues and connected to one another by linking regions of various lengths. They all possess similar ferrodoxin fold, and metal-binding motif, MXCXXC.

The need of having six metal binding domains and the manner in which they are communicating with each other is not well understood. To better understand how the last four metal binding domains function, I pursued a detailed biophysical characterization of these domains. Using molecular biology I have produced domains 3 through 6 (WLN3-6), domains 4 through 6 (WLN4-6) and domains 5 through 6 (WLN5-6). Unfolding of these constructs was performed chemically using guanidine hydrochloride (GuHCl) and monitored by circular dichroism (CD). WLN5-6 unfolds in two-state model where half of its structure is retained at 3.2 M GuHCl. However, denaturation of WLN3-6 and WLN4-6 produced a double sigmoidal curve in which the first sigmoidal transition almost matches the unfolding of WLN5-6 while the second transition matches the unfolding of WLN4. The thermal unfolding illustrated high stability for all constructs.

The unfolding of a mutant of WLN5-6 where tyrosine 48 mutated to histidine, Y532H in the whole Wilson protein, was studied by CD and femtosecond time-resolved fluorescence spectroscopy. CD showed no significant differences in the thermal and chemical unfolding but the tyrosine fluorescence was reduced because the mutation disturbs the hydrophobic core of the protein.

To study the unfolding of the protein efficiently, I introduced two-photon absorption (2PA) cross-sections as novel fluorescence technique to monitor the unfolding of proteins and imaging the change in its local electric field. Green Fluorescent Protein (GFP) was used to test our hypothesis and the method was applied in WLN5-6 after binding it to an extrinsic CPM dye. Two constructs were produced, with CPM at positions 51 and 6, WLN5-6C51_CPM and WLN5-6C6_CPM. In both constructs the anisotropy and the 2PA cross-sections increased with protein unfolding and matches the CD results, this result suggests the validity of this method to monitor protein unfolding.

Access Setting

Dissertation-Open Access

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