Download Ashworth 11-5-07

The Baker Lab - computational modeling/design of proteins
Homing endonuclease design group:
Justin Ashworth
James Havranek
Summer Thyme
Umut Ulge
target sites
computational
development
directed
evolution
computational
design
experimental
feedback
gene-targeted
specificity
Modeling homing endonuclease-DNA interactions
I-MsoI homing
endonuclease
interface
Component Energies – Arginine 72
atr rep sol dun hb res
native+min.
-7.0 0.2 3.7 3.5 -4.9 -8.2
repack+min.
-6.7 0.1 3.5 2.6 -3.9 -8.1
180°
Redesign of specificity
(proof of principle)
Experimental validation
>6400?
Binding and cleavage
Redesign of four amino acids to recognize 2
non-native basepairs results in a large,
mutually-exclusive specificity switch by
cleavage analysis.
Wildtype target
Redesigned target
[enzyme]
X-ray crystallography
The design model of this redesign was very
accurate, by comparison to the crystal structure.
(left) Rosetta model vs. Fo-Fc map;
(right) model vs. refined x-ray structure.
I-MsoI Specificity Redesigns
I-AniI Specificity Redesigns
1
28L/83R design
fraction cleaved
fraction cleaved
1
0
-7 .5
log[enzyme]
0
-5
-9
WT 5'-GCAGAACGTCGTGAGACAGTTCCG
DES 5'-GCAGAAGGTCGTGAGACCGTTCCG
log[enzyme]
-6
WT 5'-TGAGGAGGTTTCTCTGTAA
DES 5'-TAGGGAGGTTTCTCTGTAA
0.9
GCG design
fraction cleaved
fraction cleaved
1
0
-8
log[enzyme]
WT 5'-GCAGAACGTCGTGAGACAGTTCCG
DES 5'-GCAGGCGGTCGTGAGACCGCTCCG
0
-5
-9
log[enzyme]
WT 5'-TGAGGAGGTTTCTCTGTAA
DES 5'-TGAGGAGCTTTCTCTGTAA
-6
The Baker Lab - computational modeling/design of proteins
Homing endonuclease design group:
Justin Ashworth
James Havranek
Summer Thyme
Umut Ulge
target sites
computational
development
directed
evolution
computational
design
experimental
feedback
gene-targeted
specificity