A cell’s ability to recognise and respond to changes in its environment is crucial to its survival. The availability of nutrients is a fundamental part of the environment, and so cells must be able to identify when they are plentiful and when they are scarce, and adapt accordingly. GCN2 is a key protein kinase within the eukaryotic proteome, and it is activated by a drop in the intracellular concentration of amino acids. When activated, GCN2 phosphorylates the translation initiation factor eIF2, initiating the Integrated Stress Response. This causes the global inhibition of protein synthesis and the upregulation of stress response pathways. GCN2 has been implicated in a wide range of cellular processes in health and diseases, including the development of pulmonary veno-occlusive disease, neurological degeneration and cancer.

The molecular mechanisms that control the regulation and activation of GCN2 remain unclear. Some insights have been provided through genetic experiments on yeast, but the complexities of the regulatory pathways have made it difficult to decipher precise mechanistic details. For this reason, the aim of this project was to characterise the human GCN2 kinase both functionally and structurally, and to investigate the molecular mechanisms that enable it to act as a sensor of nutritional stress.

This thesis describes the development of a system to reconstitute GCN2 activation using purified components, allowing the effects of different regulators to be tested and characterised. Insights from these data alongside structural insights into the kinase allow the proposal of a model concerning how GCN2 can sense amino acid deprivation in response to various regulatory signals.

While GCN2 is activated by nutritional stress, mammalian cells have evolved a panoply of responses to environmental stress. Hsp90 is a chaperone that is required for the stability and maintenance of approximately 60 % of the human kinome, yet how it recognises client kinases is still unclear. The final chapter of this thesis describes the use of biochemical methods in combination with HDX-MS to characterise the interactions between Hsp90’s co-chaperone Cdc37 and client kinases. These analyses enabled the identification of a correlation between protein stability and dependence on Hsp90/Cdc37, and revealed that Cdc37 binding causes a dramatic conformational remodelling of the N-lobe of the kinase.