Thioredoxin homodimers: Effects of mutation and oxidation on structure and dimer formation
AuthorSanders, David A.R., 1968-
AdvisorMontfort, William R.
MetadataShow full item record
PublisherThe University of Arizona.
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.
AbstractThioredoxins are a family of small redox proteins that mediate a number of cytosolic processes in the cells of all organisms. Human thioredoxin has a number of additional functions, including the apparent stimulation of NF-κB and AP-1 transcriptional activation. It can also be exported out of the cell, where it apparently has additional functions including the ability to stimulate cell growth. The crystal structure of human thioredoxin revealed that the protein can form a homodimer. The dimer contains a disulfide bond between the Cys 73 residues of each monomer, and a novel hydrogen bond between the Asp 60 residue of each monomer that is responsible for the pH dependent behaviour of dimerization. This work was undertaken with two goals. First, three mutant protein with changes in the dimer interface, Asp58 → Ser (D58S), Ala66 → Arg (A66R) and Trp31 → Ala (W31A) were isolated and characterized. The ability of the mutant proteins to form dimers, their activities with respect to the reduction of insulin disulfides and their interaction with thioredoxin reductase were examined. The three mutations affected thioredoxins ability to form dimers: the W31A and A66R mutant proteins formed dimers less well, and the D58S mutant protein lost the pH dependence for dimer formation. Thioredoxin with a D58S mutation behaved very similarly to wild type in the activity assays, while A66R showed a 6-fold increase in K(m) and W31A showed large changes to both K(m) and V(max). From these results I suggest that the dimer interface plays a role in defining the binding site for thioredoxin reductase. The second goal was to examine the role that oxidation of the active site plays in dimerization. Oxidation, which results in structural changes to the dimer interface, also caused thioredoxin to form dimers much less readily. The reduction in dimer formation offers a possible mechanism through which thioredoxin could play a role in signaling oxidative stress. The findings detailed here begin to describe the role that the dimeric form of thioredoxin could play in physiological situations. The structural bases for the changes in dimerization are not yet fully characterized, as the monomeric form of thioredoxin has not yet been crystallized.
Degree ProgramGraduate College