To test this, we have applied our recently developed tool, Key Interactions Finder (KIF), to explore how well KIF is able to predict experimentally determined NMR hotspots (Figure ). KIF calculations were performed by comparing differences in interaction networks between the liganded and unliganded forms of the GNCA4-12 β-lactamase, that was used for determination of the NMR hotspots. As can be seen in Figure , several of the residues in the functionally important β-set (6/10) have KIF scores >0.1. Further, no residues in the α-set (which did not improve function when targeted), and only one residue in the active site set (which was already heavily optimized in our prior work ) yield large KIF residue importance scores.