Whereas the R162A mutant is completely non-functional, as previously shown in C1C2 and CrChR27,31, mutations to positively charged residues preserved the channel function, although the mutants showed significantly reduced amplitudes and impaired kinetics (Fig. 4d f and Supplementary Figure 8a d).
As shown in Fig. 5A, the K189A rNSs mutant was completely inactive whereas the D159A mutant was partially active.
Indeed, the GTPase activity of N169Q mutant was completely abolished and could not be measured (Table 2).
As in the cAMP assay, the MT1-I49N mutant was completely inactive in the ERK1/2 assay (Figure 3C).
When ΔprtV, Δlap, and ΔlapX mutants were tested in the C. elegans assay, the ΔprtV mutant was completely attenuated compared to the wild-type strain.
Furthermore, in Anabaena, a patB deletion mutant was completely defective for diazotrophic growth, but in the wildtype, its expression was restricted to heterocysts [41].
Finally, the Tn10 double mutant was completely defective for the hairpin formation step, although the kinetics of the nicking step were relatively normal (Figure 4A, F).
At both temperatures, the prkaca-F327A mutant was completely non-viable and had very low steady-state protein expression (Figure 3A).
While W542L mutant was completely devoid of enzyme activity, modifying the W542 residue with other amino acid residues partially recovered the TPPI activity.
Interestingly, when the two mutations K98A and R185A are combined, the resulting APE1 double mutant is completely devoid of its DHU-, αdA-NIR and 3'→5' exonuclease activities, indicating that these two residues are indispensable for the NIR and exonuclease functions.
