Characterisation of the conserved protein IMPACT from yeast (Yih1) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand

Abstract

Regulation of translation under conditions of amino acid starvation is an important survival mechanism to ensure the continued viability of an organism. The accumulation of uncharged tRNA under amino acid starvation conditions triggers the activation of Gcn2, a kinase that phosphorylates the translation initiation factor eIF2a, inhibiting translation initiation. The protein IMPACT has been shown to inhibit Gcn2 by sequestering Gcn1, a protein that binds Gcn2 and is required for its function in vivo. IMPACT is a highly conserved protein, but despite its conservation, little is known about the role(s) it plays in the cell. The initial aim of this study was to investigate the three dimensional structure of Yeast IMPACT Homologue 1 (Yih1) using X-ray diffraction, in the hope that knowledge of the structure would inform further understanding of its many and varied complex biological functions. Because of the difficulties in obtaining diffraction quality crystals, a number of different techniques were employed that resulted in the production of a number of different plasmids for protein expression. These included surface entropy engineering, the use of folding and stability tags, and co-crystallisation with known binding partners. Further investigation into why the protein refused to crystallise revealed an innate heterogeneity that included a propensity to bind nucleic acids. Efforts were made to determine if this was related to function without success.