alanine was fixed at position 280; alanine and threonine were allowed at position 283, and alanine, lysine and arginine at position 284. More importantly, FuncLib predicted that all variants in the available sequence space for the a-set were destabilizing, with worse Rosetta scores than the initial sequence by between 4-10 kcal mol -1 (Table and Supplementary Data Table ). Despite this, we decided to prepare and determine the Kemp eliminase activity of these variants in order to test prediction of the NMR chemical shift perturbation experiments. The five variants displayed substantially diminished Kemp elimination activity with respect to the V4 background (Figure ).