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Open Access Publications from the University of California

Structural Studies of Proteins Involved in Diseases of Protein Deposition and a Protein Involved in Nitrogenase Assembly

  • Author(s): Phillips, Aaron Hale
  • Advisor(s): Wemmer, David E
  • et al.

The work presented in this thesis describes structural investigations of proteins implicated in diseases of protein aggregation, and a protein involved in the maturation of the nitrogenase enzyme complex. Each experimental chapter is full self-contained.

Protein aggregation into plaques has been identified as a central event in many human diseases such as Alzheimer's disease, type II diabetes, and the spongiform encephalopathies. Generally, the protein plaques display a common morphology consisting of long unbranched proteinaceous fibrils commonly referred to as amyloid. The intrinsically insoluble nature of proteins contained within amyloid fibrils has greatly impeded high resolution structural studies utilizing single crystal X-ray diffraction and high-resolution liquid NMR spectroscopy. Amide hydrogen exchange is often used to probe hydrogen bonding in proteins and does not strictly require soluble protein for study. In the first chapter of the thesis, I present an amide hydrogen exchange study conducted on an amyloid forming fragment of the protein implicated in the spongiform encephalopathies, PrP(89-143). These experiments identify the strongly hydrogen bonded fibrillar core.

The next section of the thesis contains work conducted on ABeta, the amyloid forming protein implicated in Alzheimer's disease. Due to increasing lines of evidence that the ABeta fibrils themselves do not cause neurodegeneration, much effort has been directed into studying the monomeric and pre-fibrillar oligomeric states of ABeta. This work is greatly impeded by both ABeta's strong propensity to aggregate as well as the inherent heterogeneity of the molecules in solution. In order to circumvent these issues the experiments presented herein are interpreted with respect to models of the structural ensemble obtained via molecular dynamics calculations, thereby yielding a model of the structural ensemble that is validated to an extent unavailable to either technique alone.

The final section of this thesis describes work conducted on the gamma-subunit of nitrogenase, the enzyme complex responsible for the entry of nitrogen into the biosphere. The gamma-subunit's proposed role in the cell is to deliver the cofactor present at the active site of nitrogenase that is required for activity. I present biochemical experiments that demonstrate that the N-terminal domain of the gamma-subunit is responsible for mediating the interaction between the gamma-subunit and apo-nitrogenase, the solution structure of N-terminal domain of the gamma-subunit, and NMR experiments that characterize the cofactor binding site on the C-terminal domain of the gamma-subunit.

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