Connecting in vitro glutamine synthetase biophysics with the cellular environment

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Project Summary/Abstract – Connecting in vitro glutamine synthetase biophysics with the cellular environment Enzyme catalysis of vital chemical reactions sustains life. Dysregulation of these essential metabolic reactions contributes to rapid proliferation in the cancer state, devastating inherited metabolic disorders, and is involved in numerous other diseases. Contradictions between in vitro and in vivo measurements of enzyme catalysis hamper our understanding of Nature’s rules governing enzyme regulation. I propose to address these contradictions with an integrated approach using glutamine synthetase (GS), an essential metabolic enzyme, as a model. I hypothesize that integrating multiple metrics of GS function in vivo will yield more accurate functional models of GS and that modes of GS regulation will be uncovered through reconciling any differences made between orthogonal in vivo and in vitro measures of activity and composition. In my first aim, I will use cellular readouts of GS fitness and abundance multiplexed with deep mutational scanning (DMS) to elucidate the sequence determinants of GS specific activity in vivo. Next, in aim 2, I will define the thermodynamic landscape underlying GS activity as a function of oligomeric state using in vitro assays that can be compared to in vivo measurements in aim 1. Finally, I will reveal the fine conformational details that trigger GS mediated catalysis and allosteric control elicited by different oligomeric states and effectors using cryo-EM and novel kinetic assays in my third aim. Furthermore, with cryo-EM, enzyme kinetics, and oligomeric state analysis procedures in hand, those variants of interest identified from aim 1 will be fully characterized to uncover mechanisms of regulation and generate a holistic model of GS function. My in vivo specific activity metric is predicted to yield more precise information on the effect of GS variants allow for inference of allosteric networks underlying GS activity. This in vivo specific activity metric will be supported and validated by in vitro measurements. Any differences between in vitro and in vivo measurements provide the opportunity to be reconciled through additional experimentation, such as expanding in vivo assays to include unique cellular conditions. Establishing GS in vivo and in vitro connections through this approach will allow prediction of cancer somatic mutation effect on GS function. As GS occupies key nodes in vital metabolic pathways required for cell proliferation, specific inhibitors would support existing cancer therapies in GS addicted/associated cancers. Given that a tumor metabolic state is less heterogeneous than its genomic landscape, precise targeting of rouge metabolic enzyme states remains an attractive therapeutic option. Moreover, GS is a representative multimeric metabolic enzyme whose biophysical principles governing function and regulation can be compared and extended to other critical metabolic enzymes. The training I’ll receive to establish the experimental pipeline from the proposed research herein will serve me well to further expand on these regulatory principles in my career as an independent researcher at a research-intensive university.