Determination of Nutrient Ligand-Sensor Binding Affinity

Cells contain dedicated mechanisms to sense nutrient levels in the environment to regulate their growth by balancing anabolism and catabolism [1, 2]. The mechanistic Target of Rapamycin Complex 1 (mTORC1), a multi-protein kinase complex, serves as an essential growth regulator that integrates various upstream inputs including growth factors and nutrients like amino acids [1, 2] Nutrient sensors upstream of mTORC1 directly bind cognate nutrient ligands to convey their availability and thereby regulate mTORC1 signaling [1, 2]. A reliable method is needed to quantitatively determine the binding affinity (K_d) of the nutrient sensor to its ligand. In parallel, quantitative metabolomic analysis can reveal metabolite levels in fed and starved cells; which represent the physiological range of the nutrient of interest. Whether or not the binding affinity is within the physiological range serves as an indicator to determine the physiological relevance of the sensing mechanism. This chapter describes a generalizable protocol that allows reproducible determination of nutrient ligand-nutrient sensor binding affinity. Here, the S-adenosylmethionine (nutrient ligand)-SAMTOR (nutrient sensor) pair is used as an example [3]. Nutrient sensor purification, radioactive nutrient ligand incubation, and eventual scintillation counting are included, along with a description of the mathematical equation that is used to calculate the binding affinity.