C O M M U N I C A T I O N S
Figure 2. Gel mobility shift assay to determine CAP binding affinity to
33 nM lactose promoter fragment (wild-type or mutant CAP71TAG, 160
nM; buffer, 10 mM Tris, 50 mM NaCl, 500 µM EDTA, 500 µM DTT, 1
mM MgCl2, 4% glycerol, and 20 µM cAMP, pH 7.5). Lane 1, DNA only.
Lane 2, DNA + CAP wild-type. Lane 3, DNA + CAP71TAG (irradiated
at 334 nm). Lane 4, DNA + CAP71TAG (prior to irradiation at 3334 nm).
Figure 1. Absorption spectra of the mutant CAP (CAP125TAG; 50 µM)
in 50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0
buffer: red, after Ni-NTA purification and prior to irradiation; blue,
irradiation at 334 nm, 40 min; black, subsequent irradiation with 420 nm
light, 40 min.
sample was then switched back to a predominantly trans state by
irradiation at g420 nm and allowed to incubate at 4 °C. Analysis
by gel shift assays of this sample resulted in complete recovery of
transcriptional activator that regulates a number of catabolite-
sensitive operons in E. coli.10 Binding of cAMP to CAP results in
conformational changes in the protein that increase its binding
affinity to its promoter, resulting in enhanced transcription from
CAP-dependent promoters. An amber codon was introduced for
6
-1. These results
the trans form of CAP with a K of 4.0 × 10
b
M
show that AzoPhe can be used to photoregulate the binding affinity
of a transcription factor to its promoter sequence. Although complete
conversion of trans AzoPhe to the cis form cannot be obtained
due to their overlapping absorption spectra,14 these results suggest
that the genetic incorporation of AzoPhe into proteins should be
useful for temporally regulating a variety of biological processes
in vitro and in living cells.
Leu125, a residue at the dimerization interface. A C-terminal His
6
tag was added, and this mutant was expressed in rich media in the
presence of 1 mM AzoPhe, AzoPheRS, and MjtRNATyrCUA. Upon
Ni-NTA purification, about 1.5 mg of mutant CAP was obtained
per liter of cultured cells, compared to about 3 mg/L for wild-type
CAP. A UV-visible spectrum (Figure 1) of this mutant protein
shows a distinct absorbance peak at 334 nm corresponding to the
trans-azobenzene chromophore 1a. Irradiation of the mutant CAP
at 25 °C with 334 nm light led to a decrease in the 334 peak and
an increased absorbance at 420 nm, consistent with a photosta-
tionary state of approximately 45% trans-, 55% cis-azobenzene
Acknowledgment. We thank Dr. Lei Wang for guidance. We
are grateful to the DOE (ER46051) and the Skaggs Institute for
Chemical Biology for support of this work.
Supporting Information Available: Materials and methods (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
(Figure 1). The isomerized mutant was then irradiated with 420
nm light, resulting in complete conversion back to the 334 nm band.
These results show that AzoPhe can be selectively incorporated
into proteins with high fidelity and undergo reversible cis-trans
photoisomerization.
To determine whether AzoPhe could be used to photoregulate
the DNA binding affinity of CAP, AzoPhe was substituted for Ile71.
The crystal structure of the CAP-cAMP-DNA complex11 shows
that Ile71 lies in close proximity to residues Glu72, Arg82, Thr27,
and Ser128, which form an intricate network of interactions with
cAMP.12 Thus the trans and cis isomers might differentially affect
the binding affinity of cAMP to CAP and, as a consequence, the
affinity of CAP for its promoter. The mutant protein was expressed
and purified on Ni-NTA followed by FPLC purification with a
mono-S column with a gradient of 25 mM NaCl to 1 M NaCl over
References
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a 4-fold lower K
b
6
was observed for the trans CAP71AzoPhe mutant
(
9) (a) Santoro, S.; Wang, L.; Herberich, B.; King, D. S.; Schultz, P. G. Nat.
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-1
7
-1
(
K
b
∼ 4.0 × 10 M ) compared to wt CAP (K
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∼ 1.6 × 10 M
)
in the presence of cAMP (20 µM). Following photoirradiation at
6
(10) (a) Busby, S.; Ebright, R. H. J. Mol. Biol. 1999, 293, 199-213. (b)
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3
M
34 nm, the K
b
of the mutant CAP decreased 4-fold to 1.0 × 10
-1
. This is consistent with a photostationary state of 50% cis, in
(
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9
1
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(
S4), which demonstrated at the protein and cAMP concentration
used in this study that wt CAP binds roughly twice as much cAMP
as the trans CAP71AzoPhe, which binds around 40% less cAMP
upon trans-to-cis isomerization. The isomerized CAP71AzoPhe
2
1
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