Chemistry and Chemical Biology ETDs

Tyrel Bryan

Publication Date

9-12-2014

Abstract

The Haloacid Dehalogenase Superfamily (HADSF) is a ubiquitous family of enzyme with more than 32,000 members. A variety of reactions are catalyzed by HAD members, but a majority of HAD members are phosphatases. Dephosphorlyation of organophosphate metabolites are carried out in the conserved Rossmann-like core domain. The Rossman-like core domain houses the conserved active site residues necessary for catalysis and also supports domain inserts associated with substrate binding. This dissertation will focus primarily on investigating fold fitness and domain dynamics of HAD proteins more specifically three C2 capped proteins. The first part will focus on whether or not the prevailing tenet that Catalytic Function Comes at the Expense of Protein Fold Stability is applicable to the HADSF phosphatase. To test function at the cost of stability site directed mutagenesis was used to replace catalytic residues and used an array of techniques (CD/Fluorescence/SAXS) to monitored denaturation to evaluate stability in the resulting mutants. From denature results we conclude that although most catalytic residues play no role in stability, good or bad, one in particular Asp2 of DXD squiggle shows to be necessary for not only activity but stability as well. To identify what about the residue is essential to stability the side chain and interacting residues were examined. The Rossmann-like catalytic core domain is a robust domain, and to test the co-evolution of the cap and catalytic domains as a coupled folding and catalytic units will be examined. The structural co-dependency was tested by creating truncations of the catalytic and cap domains and identifying stability and activity of each domain and chimeric C2 capped proteins. Thermal denature of truncations and chimeric proteins reveal stability of each domain separately while the chimeric stability establish a robust core Rossmann-like catalytic domain. As previously discussed the cap domain increases function by increasing recognition of substrates. To examine the cap domains role in substrate-induced-fit we examined changes in protein solution conformation using small angle X-ray scattering (SAXS). Because HAD proteins have evolved separate domains for catalysis and recognition SAXS will also be used to investigate changes in conformation change due to changes in domain-domain interfacing residues. Solution scattering of Bt4131 help demonstrate an induced-fit mechanism for HAD proteins. In the presence of VO3 (phosphate analog) we see closure of cap domain whereas sugar moiety does not initiate closure of domain.

Language

English

Keywords

Protein Folding

Document Type

Dissertation

Degree Name

Chemistry

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

Allen, Karen

Second Committee Member

Mariano, Patrick

Third Committee Member

Melancon, Charles

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