As described by by Korendovych and colleagues, we quantified chemical shift perturbations by calculating Z-scores from the differences in the 1 HN and 15 N chemical shifts of the GNCA4-12 Kemp eliminase upon binding the transition-state analogue (see the Experimental Section for details). This requires the unequivocal assignment of all the cross-peaks present in the 2D 1 H, N HSQC spectrum of the free GNCA4-12 Kemp eliminase (see Figure ) followed by experiments in the presence of increasing amounts of the transition state analogue. In the case of the unligated GNCA4-12 Kemp eliminase, these assignments were done by comparison with the related and previously assigned GNCAMP Kemp eliminase variant combined with the analysis of triple resonance experiments (3D HNCO and 3D HNCA, see the Experimental Section). In this way, the chemical shifts for the backbone 1 HN, 15 N, C' and Ca atoms (listed in Table ) were obtained for about 78% of the protein residues, except for the 12 prolines, the 4 N-terminal residues and 5 C-terminal residues. Cross-peaks for the latter terminal residues could not be unequivocally identified likely because signal overlap and/or broadening or disappearance caused by exchange of the 1 HN amide protons with the water. The helices and strands deduced from the chemical shifts are in concordance with those present in the crystalline structure of the GNCAMP variant 28 (Figure ), supporting that the GNCA4-12 variant maintains the structure characteristic of our previous Kemp eliminases based on ancestral lactamase scaffolds. Representative example of perturbation of 1 H, N HSQC spectra upon transition-state analogue binding. The spectrum of unliganded GNCA4-12 is shown (black contours), together with spectra of GNCA4-12 in the presence of the transition-state analogue at the following protein/analogue ratios: 1:0.1, 1:0.14; 1:0.2; 1:1.1 and 1:2.1 (red contours). Some cross-peaks are labelled. The strong perturbation of the cross-peak assigned to A248 is visually apparent. Upon stepwise addition of the transition state analogue, most cross-peaks in the 2D 1 H, N-HSQC spectra of GNCA4-12 either remain unaffected or slightly shifted, having CSP Z-scores lower than unity. A number of cross-peaks were gradually shifted, as illustrated with the residue A248 in Figure . Such gradual shifts indicate that the binding of the transition state analogue occurs in the fast exchange regime. CSP-Z scores can be determined and unequivocally attributed to specific residues and thus to specific locations in the 3D-structure, provided that the cross-peaks are assigned in both the free GNCA4-12 and GNCA4-12 with the bound transition-state analogue.