Protein-based Artificial Enzymes - Artificial Enzymes - page 112

5.2.3
OP Nuclease Design by Mutagenesis and Chemical Modification

An additional approach to artificial nuclease design is cysteine mutagenesis followed
by conjugation with the desired catalytic group. This avoids the need to convert amino
acid residues into reactive thiols, while allowing precise control over the placement of
the reagent within the DNA-binding protein. Sigman and co-workers applied this strat-
egy to create an artificial nuclease using the bacteriophage Cro protein [26]. When

complexed with DNA, the C-terminal arm binds within the minor groove and is close
to the C-1 hydrogen of the deoxyribose on either DNA strand [26-30]. Therefore, an
alanine close to the C-terminus was mutated to a cysteine and derivatized with IOP.
After incubation with the Cro A66C-OP conjugate, 40% of the 17 base pair OR-3
operator site was cleaved within 10 min [11]. Thus, placement of the phenanthroline
group close to the DNA substrate ensured efficient nuclease activity.
Recently, the carboxy terminal domain of NarL, NarL C , was modified with 1,10-phe-
nanthroline. NarL is a response regulatory protein of E. coli and binds to a heptameric
consensus sequence [31]. Two residues of the C-terminal domain, which were mutated
and conjugated with IOP (NarL C K201C-IOP, NarL C K211C-IOP), showed high site-
specific cleavage efficiency of the top strand (Figure 5.3). When the mutated NarL C was
modified with IAOP (Figure 5.2- 2 ), a similar DNA cleavage pattern was observed on
the bottom strand. This IAOP conjugate contains a 4

k

A ˚

longer linker arm than does the

Figure 5.3 Model of the NarL C -DNA complex showing the locations
of amino acids 201 and 211. Two molecules of NarL C are positioned
above the major groove of the DNA. Color scheme: Protein secondary
structure (green), DNA (white), residue 211 (pink), residue 201
(orange).