(628c) Systematic Discovery of Protein-Metabolite Regulatory Interactions in Yeast

Metabolic engineering efforts can be hampered and reduced to trial-and-error by complications arising from uncharacterized interactions within the host organism.  These efforts would therefore benefit greatly from a systems-level understanding of metabolic regulation. Systematic characterization strategies have been successfully applied at the genomic, transcriptomic, and proteomic levels, but downstream regulatory interactions are comparatively underexplored.

A prominent example of such downstream regulatory interactions is allosteric regulation, a common phenomenon by which metabolites bind to and alter the activity of proteins. Evidence suggests that current knowledge of allosteric interactions comprises only a small fraction of all existing cases. A broad understanding of allostery in Saccharomyces cerevisiae would therefore greatly accelerate efforts to create yeast strains that produce medically and economically important compounds, and a systematic methodology for discovery and characterization is necessary to obtain this understanding.

Here, we present our work engineering higher-throughput experimental approaches to characterize and identify metabolite-protein interactions across metabolism. We have synthesized small-molecule microarrays of yeast metabolites for use as a high-throughput platform for qualitatively identifying protein-metabolite binding pairs, by probing the arrays with tagged, known proteins. These approaches have previously been applied to finding strong-affinity binders for drug leads, but have never been applied to identifying known and unknown intracellular regulatory interactions. Additionally, we have developed in vitro binding and enzymatic reaction assays coupled with gas chromatography-mass spectrometry analysis for parallel investigation of interactions in parts of the metabolome that microarrays may not effectively capture, and for validation of the regulatory function of binding hits from the array-based assay. We have demonstrated the ability to detect known binding interactions with at least nanomolar-level dissociation constants using both small-molecule microarrays and the in vitro binding assay, with work extending to detect lower-affinity interactions (micromolar-level K_Ds), and furthermore shown the utility of the in vitro reaction assay for qualitative characterizing and confirming of regulatory interactions.  The combined information on metabolite-protein interactions from these complementary approaches will facilitate the eventual construction of a systems-level mathematical model of S. cerevisiae metabolism.