Loss of Protein Stability Due to Formation of Intermolecular Disulfide Bonds Under the Effect of Oxidative Stress: Case Study of the RRM2 Domain from Neuropathological Protein TDP-43
Abstract
We have investigated the behavior of second RNA-recognition motif (RRM2) of neuropathological protein TDP-43 under the effect of oxidative stress as modeled in vitro. To accomplish this, we used an augmented version of H/D exchange experiments, 15N relaxation experiments, DLS experiments in conjunction with HSQC spectroscopy, controlled proteolysis, gel electrophoresis, and site-directed mutagenesis. Under oxidizing conditions RRM2 forms aggregate particles with a characteristic size of around 18 monomeric units. The protein chains within the aggregated particles are disulfide-bonded and destabilized. Two mechanisms are probable to have a hand in the formation of these aggregated particles. The first mechanism includes, formation of self-assembling disulfide-bonded dimers that cause some partial unfolding of the RRM2 domains. This unfolding will then allow for aggregation. The second mechanism entails that the RRM2 domains maintain their structural integrity when linked as disulfide-bonded dimers, but undergo a 'unfolding upon binding' moiety when the self-association of the dimers occurs. As the oxidative environment is reversed and the sample is placed under a reducing environment, the equilibrium shifts toward the monomeric form of RRM2. However, a significant proportion of protein remains sequestered in the aggregate particles. Due to the structural disorder in the aggregated particles, the particles remain highly susceptible to proteolytic cleavage. The developing representation provides some detailed awareness on what role oxidative stress can play with regard to neurodegenerative diseases. Such roles could allow for unfolding, aggregation, and proteolysis as different facets of the process.
Degree
Ph.D.
Advisors
Skrynnikov, Purdue University.
Subject Area
Molecular biology|Chemistry|Biochemistry
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