Out of 256 non-proline amino acid residues present in the GNCA4-12 scaffold (note that prolines are not observable in 1 H, N HSQC spectra), a total of 189 CSP Z-scores could be unequivocally identified. Their position in the GNCA4-12 sequence is shown in Figure . 16 of these assigned Z scores were ³1 and were thus considered to identify potential catalysis hotspots. Hence, we decided to focus experimental protein engineering to these 16 positions.