Thermal stability of I-AniI variants

1. Thermal stability of I-AniI variants Y2 : F13Y / S111Y M5 : F13Y / I55V / F91I / S92T / S111Y M5 I-AniI is less stable than WT I-AniI and Y2 variant.

2. In vitro cleavage assay WT AniI site LIB4 site I-AniI variants displayed higher cleavage activity against WT AniI site than WT enzyme, but showed the same cleavage efficiency against LIB4 site.

3. Binding assay by ITC Substrate : WT AniI site Y2 I-AniI WT I-AniI

4. Binding specificity Y2 I-AniI WT I-AniI Relative binding affinity TG AG GAGGT T T C T C T G T A A A TG AG GAGGT T T C T C T G T A A A WT I-AniI and Y2 variant showed similar binding profile against the target site with single mismatches, except for the central 4 base positions.

5. WT I-AniI Y2 I-AniI M5 I-AniI Thermal stability + + ± + (WT) ++ (LIB4) ++ (WT) ++ (LIB4) ++ (WT) ++ (LIB4) Cleavage activity + (WT) ++ (LIB4) ++ (WT) ++ (LIB4) Binding affinity N/A Binding specificity + + N/A - (WT) - (LIB4) ++ (WT) ++ (WT) Cleavage efficiency in bacteria Another property might contribute to the in vivo cleavage activity.

6. Cleavage activity at various temperatures I-AniI variants showed better cleavage efficiency below 37 °C than WT I-AniI.

7. Why do I-AniI variants possess stronger activity? F13Y might influence hydrophibic network to stabilize the protein core fold. This would confer the flexibility with the instability to the first LAGLIDADG motif. S111Y would make a novel contact with a DNA backbone. S111 Y111

8. Why do I-AniI variants possess stronger activity? I55V might make a new a contact with a DNA backbone (?). F91I might influence the hydrophobic interactions with residues on the second LAGLIDADG motif. S92T might cause a steric clash between C of T92 and L177. L177

9. Crystallization of Y2 I-Ani with WT AniI site Hopefully, coming soon.