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.
In vitro cleavage assay
WT AniIsite
LIB4site
I-AniI variants displayed higher cleavage activity against WT AniI site than WT enzyme,
but showed the same cleavage efficiency against LIB4 site.
Binding assay by ITC
Enzyme Targ et site
KD (nM)
H (kcal/mol)
S (cal/mol/deg)
-T S (kcal/mol)
G (kcal/mol)
WT I-AniI WT AniI 96 ± 10 11.2 ± 1.1 69.3 ± 3 - 21.0 - 9.8 WT I-AniI LIB4 8 ± 1 6.6 ± 0.7 59.0 ± 3 - 17.9 - 11.3 Y2 I-AniI WT AniI 11.6 ± 3.0 17.1 ± 1.0 93.0 ± 3.1 - 28.2 - 11.0 Y2 I-AniI LIB4 13.9 ± 2.0 14.8 ± 2.4 84.7 ± 7.6 - 25.7 - 10.9
WT I-AniI Y2 I-AniI
Substrate : WT AniI site
Binding specificity
WT I-AniI Y2 I-AniI
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10
A C G T
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10
T G A G G A G G T T T C T C T G T A A A T G A G G A G G T T T C T C T G T A A A
Re
lativ
e b
ind
ing
aff
inity
WT I-AniI and Y2 variant showed similar binding profile against the target site with single
mismatches, except for the central 4 base positions.
WT I-AniI Y2 I-AniI M5 I-AniI
Thermal stability + + ±
Cleavage activity+ (WT)
++ (LIB4)++ (WT)++ (LIB4)
++ (WT)++ (LIB4)
Binding affinity+ (WT)
++ (LIB4)++ (WT)++ (LIB4)
N/A
Binding specificity + + N/A
Cleavage efficiency in bacteria- (WT)
- (LIB4)++ (WT) ++ (WT)
Another property might contribute to the in vivo cleavage activity.
Cleavage activity at various temperatures
I-AniI variants showed better cleavage efficiency below 37 °C than WT I-AniI.
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.
S111Y111
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
Crystallization of Y2 I-Ani with WT AniI site
Hopefully, coming soon.
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