Characterizing glucokinase variant mechanisms using a multiplexed abundance assay

Abstract

Amino acid substitutions can perturb protein activity in multiple ways. Understanding 14 their mechanistic basis may pinpoint how residues contribute to protein function. Here, we 15 characterize the mechanisms of human glucokinase (GCK) variants, building on our previous 16 comprehensive study on GCK variant activity. We assayed the abundance of 95% of GCK 17 missense and nonsense variants, and found that 43% of hypoactive variants have a decreased 18 cellular abundance. By combining our abundance scores with predictions of protein 19 thermodynamic stability, we identify residues important for GCK metabolic stability and 20 conformational dynamics. These residues could be targeted to modulate GCK activity, and 21 thereby affect glucose homeostasis.