UCSF

Determining how the sequence of a protein effects its function and physical properties builds the base of a foundation needed to investigate how a protein interacts with substrates and other proteins, to predict the functional effects of variants, understand disease phenotypes, and to improve rational drug and protein design. In my thesis, I describe contributions to understanding how we can understand the molecular basis for nonsynonymous disease mutations in the human metabolic enzyme glutamine synthetase and how we can leverage both experimental and computational approaches to find secondary mutations that can rescue the functional defects of a disease variant, termed compensatory mutations. In chapter one, I developed a computational pipeline to identify potential protein targets in which a human disease mutation is present as the wild type residue in other genetic contexts, called compensated pathogenic deviations (CPD), as well as identify potential compensatory sites in those protein targets. I then chose one target, the human metabolic enzyme glutamine synthetase (GS), and experimentally tested every possible single secondary mutation in the background of the clinically validated R341C disease variant to map the compensatory landscape. We found several sites of compensation that were able to rescue the defects induced by R341C. Interestingly, most of them were far from both the active site and the residue 341, pointing to a potentially more complex method of compensation involving long-range interactions or alteration of the conformational landscape of GS. In chapter two, we aimed investigated if human glutamine synthetase (hGS) activity was regulated by formation of higher order oligomeric assemblies, as is common with some metabolic enzymes, and if this balance could be biased through addition of metal cofactors or through mutation. There has been previous evidence that GS in both e.coli and s. cerevisiae form filamentous assemblies, with the ring structure stacking back-to-back in times of cellular stress as a way to store catalytic potential. We utilized to complementary structural methods to visualize and quantify the oligomeric form of hGS: negative stain electron microscopy and size exclusion chromatography coupled small angle x-ray scattering (SEC-SAXS). We perturbed hGS by increasing the concentration of Mg2+ or Mn2+, varying pH, and single amino acid mutation (including the hGS disease variants) and saw no evidence of higher order oligomers. We did however observe an equilibrium between the pentamer and decamer that existed across all conditions, with the equilibrium shifted more towards the pentamer state than previously expected. While we have concluded that hGS does not regulate catalytic activity through formation of filaments, as do glutamine synthetases in other species, the delicate balance we probed between pentamer and decamer is useful in characterizing the biology of the native hGS and informs future purification and kinetic studies.