Chemistry Reference
In-Depth Information
signifi cantly in the sequence specifi city of the conjugates, being able to discriminate
between different DNA sequences. Furthermore, the sequence selectivity was in
many cases perturbed by a single amino acid modifi cation,
46,47
demonstrating the
versatility of peptides as systems utilized for DNA recognition. However, in some
cases there is a possibility that the intrinsic preference of the metal complex for a
specifi c DNA sequence may interfere with sequence specifi city conferred by the
peptide moiety. Hence, the resulting sequence specifi city may differ from that
expected in cases where the peptide sequence is derived from a DNA-binding
protein.
48
Although, small peptides are composed of readily available building blocks and
it is facile to tune the peptide composition to achieve the desired properties and
variations, they lack the structural constraints and control that may be needed for
predictive design. Indeed, their conformational fl exibility accounts for the failure of
recognizing the same sequence as the native protein they are derived from.
48,49
12.3
Metallointercalator - Metallopeptide Conjugates
Design of tethered peptides has also been extended to mimicking the properties of
certain proteins, such as DNA hydrolases, zinc-binding proteins and other important
cell proteins which interact with DNA. Barton and coworkers tethered a peptide
designed
de novo
, based on the active sites of metal-containing hydrolases to the
metallointercalator [Rh(phi)
2
(bpy
)]
3+
.
52
Two histidines were placed in positions 7
and 11 along a 16-residue peptide (P1) to create a zinc coordination site on one face
of an a-helix. A glutamate was included at position 4, and glutamate-lysine salt
bridges, as well as space-fi lling alanines were added to increase a - helicity (Figure
12.6). Both helical content and cleavage activity of plasmid DNA reached a maximum
at stoichiometric amounts of Zn
2+
using micromolar concentrations of the chimeric
complex, owing to the strong binding affi nity of the intercalating moiety. Further-
more, direct evidence was obtained for a hydrolytic cleavage reaction.
52
Interest-
ingly, the same peptide, when conjugated to ethidium, in the presence of Zn(II),
forms a closed chelate structure which prevents binding to DNA and concomitant
cleavage.
53
The importance of several key residues of P1 was further assessed by determin-
ing the DNA cleavage effi ciency of mutations in plasmid cleavage assays.
54
Not
surprisingly, the two histidine residues at positions 7 and 11 were found to be impor-
tant for zinc coordination and cleavage activity. On the other hand, the cleavage
rate constant was not affected upon mutation of the glutamic acid at position 4.
54
Also, the selection of the metallointercalator seemed to be critical for the hydrolytic
activity, since the attachment of P1 to [Rh(phen)(bpy)(chrysi)]
3+
, a metallointercala-
tor which binds to DNA mismatches, did not produce Zn
2+
- promoted hydrolysis.
54
In addition, zinc(II)-promoted cleavage of supercoiled plasmid has also been dem-
onstrated for another metallointercalator-metallopeptide conjugate bearing a short
b-hairpin derived from the
Bam
HI restriction endonuclease, incorporating the three
important catalytic residues (Asp94, Glu111 and Glu113) responsible for the hydro-
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