Characterizing glucokinase variant mechanisms using a multiplexed abundance assay

Amino acid substitutions can perturb protein activity in multiple ways. Understanding their mechanistic basis may pinpoint how residues contribute to protein function. Here, we characterize the mechanisms of human glucokinase (GCK) variants, building on our previous comprehensive study on GCK variant activity. We assayed the abundance of 95\% GCK missense and nonsense variants, and found that 59\% of hypoactive variants have a decreased cellular abundance. By combining our abundance scores with predictions of protein thermodynamic stability, we identify residues important for GCK metabolic stability and conformational dynamics. These residues could be targeted to modulate GCK activity, and thereby affect glucose homeostasis. Overall design: Three regional libraries of human glucokinase variants (aa 2-171, 172-337, 338-465) were transformed into yeast (BY4741), and were grown on SC+leucine+methionine+histidine medium with methotrexate to select for glucokinase variant abundance. The variants were sequenced before and after selection using 14 tiles spanning the glucokinase ORF to determine their change in frequency and thereby effect on protein abundance.