Chemistry Reference
In-Depth Information
iodoacetyl - 5 - amino - 1,10 - phenanthroline) was attached to the coiled - coil (CC)
dimerization region based on an artifi cial sequence developed by Hodges and cow-
orkers (Figure 12.5).
45
The DNA-binding domain of the protein was taken from the
basic region of the GCN-4 transcription factor (GBR). The photoactive metal
complex was attached to Cys29, the most heavily solvent-exposed position of the
coiled-coil closest to the DNA-binding domain, and, as indicated by its emission
spectrum, the ruthenium centre remained exposed to solvent when coupled to the
bZIP peptide. Electrophoretic mobility shift assays showed that the chimeric metal-
loprotein retained the essential DNA recognition properties of the native GCN-4
transcriptional activator. Peptide-DNA complexes were formed with the AP1 and
CRE sequences, known recognition sites of GCN-4, but not the divergent Sp1
sequence. Peptide titration studies indicated that the affi nities of (GBR-CC)Ru for
the AP1 and CRE sites were comparable. (GBR-CC)Ru did not produce observable
photoinduced DNA damage, probably due to the separation distance between the
ruthenium sites and the DNA bases.
44
The DNA recognition properties of the a
3
- helix of the DNA - binding
phage P
22
repressor were investigated by attachment onto the metallointer-
calating [Rh(phi)
2
(phen
)]
3+
(phi = 9,l0 - phenanthrenequinone diimine; phen
= 5 -
(amidoglutaryl) - 1,10 - phenanthroline).
46
Remarkably, a single glutamate at position
10 was found to be essential in directing DNA site-recognition to the sequence
5
′
′
. It is noteworthy that conservative modifi cations of this glutamate, includ-
ing aspartate substitution or derivatization to the glutamate methyl ester, abolished
the 5
′
- CCA - 3
′
recognition. On the other hand, circular dichroism indicated signifi -
cant a-helical content in the peptide, which depended upon the presence of the
glutamate. The experimental evidence indicated that the glutamate may play a dual
role, folding the metal-peptide complexes into a unique conformation and also
interacting directly with DNA. However, the metal-peptide complexes did not
preferentially target the putative operator sequence for protein P
22
R. This fact may
arise because in the native protein, intraprotein interactions determine the orienta-
tion of the a
3
recognition helix, whereas in the metal-peptide complexes, the peptide
conformation may be directed instead by the metal centre and the solvent. Further-
more, moving the glutamate at position 10 in the sequence of the appended peptide
to position 6 changed the sequence preference of the metallointercalator-peptide
conjugate to 5
′
- CCA - 3
′
, whereas replacing Glu
6
with Arg resulted in a change in
the apparent consensus sequence from 5
′
- ACA - 3
′
.
47
These results
clearly illustrate that with a single amino-acid modifi cation, the recognition sequence
and the photocleavage characteristics of the metallointercalator-peptide conjugate
can be perturbed.
A similar work by N.Y. Sardesai and J.K. Barton focused on the DNA recogni-
tion properties of metal-peptide complexes containing peptides derived from the
sequence of the a
3
recognition helix of the phage 434 repressor protein (434R)
appended to the metallointercalator [Rh(phi)
2
(phen
′
- ACA - 3
′
to 5
′
- (T/G)CA - 3
′
)]
3+
.
48
A series of peptide
sequences was designed, based upon 434R and criteria for maximizing peptide a -
helicity. The three Gln residues were left unchanged because they make base-spe-
cifi c contacts with the 434R consensus sequence 5
′
. Therefore, a family of
peptides a
A
, a
B
(a
A
- Q7R), a
C
(a
A
- Q3R), and a
D
(a
A
-Q3A) was formed, in which
peptides were different from one another by a single amino acid at one of the three
′
- ACAA - 3
′
