Biophysical studies of two key regulatory proteins of the translation initiation pathway
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Translation is the process by which proteins are synthesized on the ribosome according to the nucleotide code provided by the mRNA. This highly sophisticated multistep process is tightly regulated at many levels. One of the key mechanisms employed to regulate protein synthesis is via post-translational modifications of translation initiation factors. This dissertation describes the structures of translation initiation factors eIF2α and eIF4E; two key proteins involved in regulating the protein synthesis pathway. The Structure and Dynamics of the α subunit of eIF2 The alpha subunit of translation initiation factor 2 (eIF2α) is the target of specific kinases that can phosphorylate a conserved serine residue as part of a mechanism for regulating protein expression at the translational level in eukaryotes. The structure of the 20 kDa N-terminal region of eIF2α from Saccharomyces cerevisiae was determined by X-ray crystallography at 2.5 Å resolution. The S. cerevisiae eIF2α has a rather elongated structure that consists of a five-stranded antiparallel β-barrel in the N-terminal, followed by an almost entirely helical domain. The crystal structure of the yeast eIF2α is significant as it provides the first view of the putative phosphorylation site, serine 51. In addition to the structure, the dynamic properties of the α subunit of eIF2 were investigated in solution by NMR spectroscopy. Determination of the redox state of wheat eIF4E Translation initiation factor eIF4E initiates protein synthesis by binding to the m7GpppN cap structure of the mRNA. In this dissertation, the results of the redox studies of wheat eIF4E in solution are presented. The NMR data indicate that all the cysteine residues in wheat eIF4E are reduced in solution. However, C113 and C151 can be oxidized to form a disulfide bond in vitro, causing some structural changes in the protein. It was observed that the majority of the amino acid residues affected by this selective oxidation seem to be concentrated in the cap-binding face of the protein. Based on these observations it can be hypothesized that plants may employ changes in the redox state of conserved cysteine residues as part of a regulatory mechanism to control protein synthesis.