Fluorescence quenching and kinetic studies of
conformational changes induced by DNA and cAMP
binding to cAMP receptor protein from Escherichia coli
Magdalena Tworzydło, Agnieszka Polit, Jan Mikołajczak and Zygmunt Wasylewski
Department of Physical Biochemistry, Faculty of Biotechnology, Jagiellonian University, Krako
´
w, Poland
Cyclic AMP receptor protein (CRP), allosterically
activated by cAMP, is a multipotent transcription
regulating protein engaged in the control of more
then 100 genes in Escherichia coli [1,2]. The protein is
a homodimer. Each subunit consists of 209 amino
acid residues folded into two distinct domains. The
N-terminal domain, composed of amino acid residues
1–133, contains a cAMP-binding pocket that binds
the cAMP in the anti conformation. The N-terminal
domain is coupled with the C-terminal domain by a
flexible hinge region made up of residues 134–138.
The smaller, C-terminal domain possesses amino acid
residues 139–209 and contains the helix-turn-helix
(HTH) motif. The crystal structure of the CRP–DNA
complex revealed the existence of a second site
between the hinge and the turn of the HTH where
cAMP is bound in the syn conformation [3]. Upon
cAMP binding in the anti conformation, CRP under-
goes allosteric conformational changes that enable the
protein to recognize specific DNA sequences [2,4].
Therefore, it has been suggested that CRP can exist
in solution in at least three conformational states,
Keywords
cAMP receptor protein (CRP); CRP–DNA

¨
rster resonance energy transfer between
labeled Cys178 of CRP and fluorescently labeled DNA sequences to study
the kinetics of DNA–CRP interactions. The results thus obtained lead to
the conclusion that CRP can exist in several conformational states and that
their distribution is affected by binding of both the cAMP and of specific
DNA sequences.
Abbreviations
CRP, cyclic AMP receptor protein; CRP–AEDANS, CRP covalently labeled with 1,5-I-AEDANS attached to Cys178; apo–CRP, unligated CRP;
FRET, Fo
¨
rster resonance energy transfer; FQRS, fluorescence-quenching-resolved spectra; galF, a fragment of DNA sequence recognized by
CRP in the galP1 promoter covalently labeled with fluorescein at the 5¢ end; HTH, helix-turn-helix; lacF, a fragment of DNA sequence
recognized by CRP in the lacP1 promoter covalently labeled with fluorescein at the 5¢ end; ICAPF, consensus DNA sequence recognized by
CRP covalently labeled with fluorescein at the 5¢-end; wt, wild type.
FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS 1103
i.e. free CRP, CRP–(cAMP)
2
and CRP–(cAMP)
4
.In
the presence of % 100 lm cAMP, the protein becomes
activated by the formation of a CRP–(cAMP)
2
com-
plex and it is then able to recognize and bind specific
DNA sequences and stimulate transcription [5].
Unfortunately, the crystal structure of unligated CRP
has not yet been established, which makes a simple
comparison between the two forms of the protein

Depending on the location of the CRP-binding site
on the DNA promoter and the mechanism of CRP–
RNA polymerase interaction, the simple CRP-depend-
ent promoters are divided into two classes [1]. Class I
promoters, such as lacP1, are characterized by the
location of the CRP-binding site centred at position
)61.5. In the case of class II promoters, such as galP1,
the CRP-binding site is located at position )41.5. The
activation of the transcription process requires the
interaction between the RNA polymerase a subunit
C-terminal domain and the CRP-activating region,
AR1 [9]. The class II promoter requires the interaction
with both the AR1 activation region of CRP and
the activation region of AR2, located in the CRP
N-terminal domain [10].
Each CRP subunit contains two tryptophan residues
at positions 13 and 85 (Fig. 1), both located in the
protein’s N-terminal domain [11]. Trp85 is located
near the anticAMP-binding site and Trp13 is situated
close to the activation region, AR2, of CRP. Using
single tryptophan-containing mutants, we have recently
shown that the binding of cAMP in the CRP–(cAMP)
2
complex alters the surroundings of Trp13, whereas
its binding in the CRP–(cAMP)
4
complex leads to
changes in the Trp85 microenvironment [7]. We
present evidence that CRP binding to the different
DNA sequences leads to long-distance conformational

Fig. 1. Structure of the cyclic AMP receptor protein (CRP) dimer
complexed with DNA. The locations of tryptophan residues are
marked in red, the location of the Cys178 residue is indicated in
yellow and fluorescein is shown in green. The figure was generated
by
WEBLAB VIEWERPRO (version 3.7) using atomic coordinates for the
cAMP–CRP–DNA complex [44]. The coordinates were obtained
from the Brookhaven Protein Data Bank (accession code 1CGP).
CRP conformational changes induced by DNA and cAMP M. Tworzydło et al.
1104 FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS
Results
Steady-state fluorescence quenching studies
The fluorescence quenching studies with iodide and
acrylamide were performed in 20 mm Tris⁄ HCl buffer,
pH 7.9, containing 0.1 m NaCl and 0.1 mm EDTA. In
measurements involving the protein–ligand complex,
the final concentration of cAMP was 100 lm. In all
cases, the excitation wavelength was 295 nm, so it can
be assumed that the fluorescence emission observed
was only from tryptophan residues.
A typical Stern–Volmer plot of fluorescence quench-
ing of the single tryptophan of the CRPW85A mutant
is shown in Fig. 2. The downward curvature of the
plot indicates the presence of two or more emitting
components which differ in a Stern–Volmer quenching
constant, K
SV
. The fluorescence quenching data were
analyzed according to Eqn (3), by using a nonlinear
least-squares procedure. The analysis was conducted

SV1
value from 9.61 m
)1
to
10.08 m
)1
and the more visible increase of the K
SV2
value from 1.69 m
)1
to 2.85 m
)1
.
When acrylamide was used as a quencher, the
Stern–Volmer plots of CRPW85A and its complex
with cAMP showed a small upward curvature indica-
ting that a static quenching mechanism is involved
(Fig. 2B). For both species, the best fits were obtained
for a model in which one component is accessible to
the nonionic quencher. For CRPW85A, the acrylamide
Stern–Volmer constant is equal to 5.76 m
)1
, while for
the cAMP complex, K
SV
¼ 6.62 m
)1
, and the values of
a static quenching constant, V, are 0.84 m
)1

1
¼ 0.48, K
SV2
¼ 2.89 M
)1
,
f
2
¼ 0.40. (B) Typical Stern–Volmer plots for acrylamide quenching
of CRPW85A (
) and of CRPW85A–(cAMP)
2
(h). The solid lines
represent the best fits with the following parameters: CRPW85A,
K
SV
¼ 5.64 M
)1
, V ¼ 0.74 M
)1
, f ¼ 1; CRPW85A–(cAMP)
2
, K
SV
¼
6.45
M
)1
, V ¼ 0.22 M
)1

the fluorescence emission of the complexes. The high-
est Stern–Volmer constant, amounting to 7.45 m
)1
,
characterizes the CRPW85A–ICAP complex. For
CRPW85A–lac, the value of K
SV1
is 5.54 m
)1
, and for
CRPW85A–gal, the value of K
SV2
is 5.02 m
)1
. The
quenched components account for % 78–81% of the
total fluorescence emission.
When acrylamide was used for quenching, the
Stern–Volmer plots for two complexes of CRPW85A,
with ICAP and lac sequences, were found to be
linear so the model with one totally quenched com-
ponent was used for calculations. The dynamic
quenching constant values for these two species
were 6.35 and 6.15 m
)1
, respectively. Only for the
CRPW85A–gal complex did the upward curvature
appear, indicating the presence of static quenching,
characterized by the constant V ¼ 1.35 m
)1

m
¼ Sf
i
s,
are presented in Table 2.
Table 1. Fluorescence quenching parameters for CRPW85A, CRPW85A–(cAMP)
2
and CRPW85A–DNA complexes. Iodide and acrylamide
quenching studies were performed in Tris buffer, pH 7.9 at 20 °C. In the experiments with CRPW85A complexed to cAMP and DNA, the
concentration of cAMP was 100 l
M. Quenching data were fitted to either a one- or a two-component model (Eqn 1). The presented parame-
ters were obtained for the model characterized by minimum values of reduced v
2
. K
SV
and V are average values calculated for the wave-
length range between 330 and 370 nm. The error did not exceed 5%. FQRS, fluorescence-quenching-resolved spectra.
Species K
SV1
(M
)1
) K
SV2
(M
)1
) V (M
)1
) f
1
FQRS

acceptors.
The application of the FRET method allowed us to
obtain more information about the binding process
between protein and DNA. One of the advantages is
the possibility of determining the kinetics of the associ-
ation by monitoring the time course of the FRET
effect. Using fluorescein-labeled DNA as the acceptor,
we observed a small increase in acceptor fluorescence
but a significant decrease in IAEDANS emission.
Quenching of the IAEDANS fluorescence intensities is
not solely governed by Fo
¨
rster nonradiative energy
transfer in the CRP–DNA complex, but also by the
DNA itself. The addition of unlabeled DNA to CRP–
AEDANS significantly decreased the fluorescence
intensities of the dye (data not shown) and therefore we
decided to use the acceptor fluorescence to monitor the
CRP–DNA interaction in the FRET kinetic measure-
ments. Mixing an IAEDANS-labeled CRP with a fluo-
rescein-labeled oligonucleotide resulted in an increase
of % 7% in the acceptor fluorescence at the donor exci-
tation wavelength, reaching a plateau at % 0.3 s.
For all DNA sequences and CRP concentrations,
the kinetic traces could be fitted well by a single-expo-
nential curve. The plots of the inverse time constant
(k
obs
) are linear (Fig. 9) and the values of k
off

¼ 0.43.
Fig. 3. Fluorescence-quenching-resolved spectra (FQRS) of
CRPW85A with excitation at 295 nm. Iodide was used as a quen-
cher. The upper panel represents a plot of Stern–Volmer constants
as a function of the emission wavelength. The lower panel shows
the FQRS spectra: (
) the total emission spectrum with a maxi-
mum at about 342 nm; (
) the more quenchable component with a
maximum at about 350 nm, characterized by an average value of
K
SV1
¼ 9.61 M
)1
and a fraction f
1
¼ 0.55; and ( ) the less quencha-
ble component with a maximum at about 338 nm, characterized by
an average value of K
SV2
¼ 1.69 M
)1
and a fraction f
2
¼ 0.45.
M. Tworzydło et al. CRP conformational changes induced by DNA and cAMP
FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS 1107
CRPÀðcAMPÞ
2
þ DNA À!

Association constants (K
a
) of CRP with the three
investigated sequences of DNA – lacF, galF and
ICAPF – are summarized in Table 3.
Discussion
The molecular mechanism of signal transduction
within CRP upon binding of the allosteric inductor to
CRP high-affinity binding sites involves a sequence
of protein conformational changes, which shift the
protein from a low-affinity nonspecific DNA-binding
protein to a state of the protein that binds DNA with
Fig. 5. (A) Typical Stern–Volmer plots for iodide quenching of
CRPW85A complexes with DNA. The solid lines represent the best
fits with the following parameters: (r) CRPW85A–ICAP, K
SV1
¼
6.57
M
)1
, f
1
¼ 0.80; (d) CRPW85A–lac, K
SV1
¼ 5.46 M
)1
, f
1
¼ 0.78;
and (,) CRPW85A–gal, K

shows the FQRS: (e) the total emission spectrum with maximum
at % 340 nm; (r) the quenchable component with a maximum at
% 345 nm, characterized by an average value of K
SV1
¼ 7.45 M
)1
and a fraction f
1
¼ 0.80; and ( ) the unquenchable component with
a maximum at %338 nm, characterized by an average value of
K
SV2
¼ 0.00 M
)1
and a fraction f
2
¼ 0.20.
CRP conformational changes induced by DNA and cAMP M. Tworzydło et al.
1108 FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS
high affinity and sequence specificity [2]. A variety of
biochemical and biophysical studies [13–16], including
our fast-kinetics studies [17,18], as well as steady-state
and time-resolved fluorescence [7,19] investigations,
have shown that the allosteric mechanism involves sub-
unit realignment and hinge reorientation between the
domains. Our previous FRET measurements have
shown that cAMP binding to the anti sites of CRP
shifts the average distance from the C-terminal domain
towards the N-terminal domain from 26.6 A
˚

Fig. 7. Fluorescence-quenching-resolved spectra (FQRS) of
CRPW85A–lac with excitation at 295 nm. Iodide was used as a
quencher. The upper panel represents a plot of Stern–Volmer con-
stant as a function of the emission wavelength. The lower panel
shows the FQRS spectra: (s) the total emission spectrum with a
maximum at % 340 nm; (d) the quenchable component with a
maximum at % 346 nm, characterized by an average value of
K
SV1
¼ 5.54 M
)1
and a fraction f
1
¼ 0.78; and ( ) the unquen-
chable component with a maximum at % 340 nm, characterized by
an average value of K
SV2
¼ 0.00 M
)1
and a fraction f
2
¼ 0.22.
Fig. 8. Fluorescence-quenching-resolved spectra (FQRS) of
CRPW85A–gal with excitation at 295 nm. Iodide was used as a
quencher. The upper panel represents a plot of Stern–Volmer con-
stant as a function of the emission wavelength. The lower panel
shows the FQRS: (,) the total emission spectrum with a maximum
at about 340 nm; (.) the quenchable component with a maximum
at about 343 nm, characterized by an average value of K
SV1

obtain a single tryptophan-containing mutant protein,
which will allow for a more straightforward interpret-
ation of fluorescence quenching data.
In this study, we used site-directed mutagenesis to
obtain the CRPW85A mutant and used the FQRS
method to observe conformational changes in the pro-
tein upon binding of cAMP and fragments of DNA
possessing specific sequences. Each CRP wild-type
(CRPwt) subunit contains two tryptophan residues at
positions 13 and 85, both located in the N-terminal
domain of the protein [11,23]. Our previous fluores-
cence quenching investigations [24] of CRPwt have
shown that in apo–CRP, % 80% of the tryptophan
fluorescence emission can be attributed to Trp13 and
20% of the fluorescence emission originates from
Trp85. Our recently presented data concerning CRP
mutants containing a single Trp13 or Trp85 residue
indicate that binding of cAMP to anti sites in the
CRP–(cAMP)
2
complex leads to changes in the Trp13
microenvironment, whereas its binding to syn sites in
the CRP–(cAMP)
4
complex alters the surroundings of
Trp85 [7].
The results presented in this report provide further
evidence that binding of cAMP to the anti site of CRP
induces local structural changes in the vicinity of
Table 2. Fluorescence lifetimes and bimolecular quenching constants values for CRPW85A, CRPW85A–(cAMP)

)1
Æs
)1
)
x10
)1
k
q1
(M
)1
Æs
)1
)
x10
)1
CRPW85A 3.09 0.69 0.58 2.31 4.16 0.73 2.49
CRPW85A–(cAMP)
2
2.99 0.65 0.55 2.14 4.67 1.23 3.09
CRPW85A–ICAP 2.54 0.59 0.29 1.62 4.60 – 3.92
CRPW85A–lac 4.23 0.57 0.62 2.68 2.07 – 2.29
CRPW85A–gal 3.93 0.51 0.62 2.31 2.17 – 3.39
Fig. 9. Kinetics of binding between IAEDANS-labeled CRP and fluo-
rescein-labeled DNA, as measured by stopped-flow fluorymetry of
the Fo
¨
rster resonance energy transfer (FRET). Measurements were
performed at 20 °C, in buffer B, pH 8.0, with a DNA concentration
of 0.2 l
M:(d) lacF;(,) galF;(r) ICAPF. Excitation was at 340 nm

CRPwt–galF 5.1 ± 0.9 2.4 ± 0.2 4.7 ± 0.9
CRP conformational changes induced by DNA and cAMP M. Tworzydło et al.
1110 FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS
Trp13. Our fluorescence quenching measurements of
apo–CRPW85A with iodide demonstrate that the
steady-state fluorescence spectra of Trp13 can be
resolved into two components by using the FQRS
method. This result clearly shows that CRP exists in
two distinct conformational states, each of which is
characterized by a different microenvironment of
Trp13. One of these states is characterized by its own
fluorescence emission spectra with a maximum at
350 nm and the second state is characterized by a
maximum emission spectrum at 338 nm. These two
forms of the protein account for 55% and 45% of the
total fluorescence emission, respectively. In contrast to
the Trp13 residue, the tryptophan located at position
85 is characterized by one distinct fluorescence spec-
trum (data not shown). The conformational state of
apo–CRP, which possesses a maximum of the fluores-
cence emission spectrum at 350 nm, can be character-
ized by a Trp13 Stern–Volmer quenching constant,
K
SV
¼ 9.6 m
)1
. If the average lifetime of Trp13 is
assumed to be 2.3 ns, then the bimolecular rate-
quenching constant, k
q

m
)1
Æs
)1
, is almost half
that of the iodide rate-quenching constant. It has been
well documented that nonionic acrylamide can penet-
rate into the matrix of globular protein by diffusion,
which is facilitated by small-amplitude fluctuations in
the protein structure [25,26]. The process of quenching
the fluorescence of Trp residues in protein by acryl-
amide is more effective than by using the iodide ion
[25,26].
Resolving the component spectra of the Trp13 resi-
due of CRPW85A by using the FQRS method and
fluorescence lifetime measurements enabled us to com-
pare the fractional contributions of the fluorescence of
the red and blue components from the solute quench-
ing experiments by using the fractional contributions
of the short and long lifetimes of the Trp13 residue
obtained by lifetime measurements. A comparison of
the fractional contribution values presented in Tables 1
and 2 shows a significant discrepancy, which suggests
that the two Trp13 residues present in the CRPW85A
homodimer do not fluoresce independently and that
there is an energy transfer between them. A similar
observation has been drawn from the resolved fluores-
cence lifetime and solute quenching measurements per-
formed for several two-tryptophan-containing proteins
[27]. It may also be supposed that the fluorescence

complex, is % 25.5 A
˚
[6],
the observed changes in Trp13 fluorescence quenching
by iodide and acrylamide result from the transduction
of the conformational changes in the protein moiety
and increase the dynamic motion around the Trp13
residue. This observation is in congruence with our
previous time-resolved anisotropy fluorescence meas-
urements of CRP, which show that cAMP binding to
the protein leads to an increase in the structural
dynamic motion around Trp13 [7]. As the Trp13 resi-
due is close to the activation region of CRP, AR2,
which is responsible for the interaction of the protein
with the a subunit of RNA polymerase, it may be
argued that the changes in the CRP dynamics in this
molecule region can play an important role in signal
transmission in the protein. Similarly, it has been
shown that the Trp13 residue in CRP is directly
engaged in the formation of the CRP complex with
another gene-regulatory protein, such as CytR, in the
CRP–CytR–DNA complex [28].
It is well established that the CRP allosteric activa-
tion involves conformational changes that are trans-
mitted from the N-terminal domain to the C-terminal
domain of the protein and, in consequence, enable
CRP to recognize the specific DNA sequences [2,4,11].
The results presented in this work provide evi-
dence for conformational signal transduction in the
M. Tworzydło et al. CRP conformational changes induced by DNA and cAMP

tribution, have been obtained for two CRP states: one
with an iodide-quenchable and the second with an
iodide-unquenchable Trp13 residue. Binding DNA
sequences to CRP causes only a small change in the
maximum of the two resolved fluorescence emission
spectra, but shows that the iodide-quenchable compo-
nents account for % 75% of the total emission of
Trp13, in comparison to % 55% in the CRP–(cAMP)
2
complex (Table 1). As the binding of the tested DNA
sequences also leads to changes in the average fluores-
cence lifetime of Trp13, it may be expected that the
observed changes result from both the static and
dynamic processes that occur in the microenviron-
ments of this residue. Thr10, Asp109 and His17, which
are located within a distance up to 5 A
˚
[29] are the
most probable candidates as quenching residues of
CRP, in the vicinity of Trp13. The accessibility for
iodide as well as acrylamide, expressed by k
q
values
(Table 2), differs for the three studied DNA sequences
and clearly shows that binding of the particular DNA
to CRP causes different local changes in Trp13 residue
exposition. As this residue is located close to the acti-
vation region, AR2, which is responsible for the inter-
action with the RNA polymerase, it is tempting to
suggest that the binding of CRP to the DNA promoter

m
)1
Æs
)1
and 2.4 · 10
6
m
)1
Æs
)1
, determined for
ICAP, lac and gal, respectively, are very similar to the
values of rate constants calculated for the interaction
of DNA with other proteins [32–34]. However, the
monomolecular dissociation rate constants determined
for the CRP–ICAP, CRP–lac and CRP–gal complexes,
of 5.8 s
)1
, 8.5 s
)1
and 5.1 s
)1
, respectively, are signifi-
cantly higher than the range between 10
)3
and 10
)2
s
)1
that has been found for other proteins which interact

5
m
)1
for ICAP, lac and gal,
respectively. These values are slightly lower than the
association constants of 4.0 · 10
5
m
)1
and 11.1 ·
10
5
m
)1
that were determined by isothermal titration
calorimetry for lac and gal, respectively [35]. The
26 bp long DNA sequences – lac, gal and ICAP – have
CRP conformational changes induced by DNA and cAMP M. Tworzydło et al.
1112 FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS
almost all the DNA determinants required for the
maximum affinity for CRP–DNA interactions [36].
The sequence ICAP is palindromic and this may be
responsible for the fact that higher values of associ-
ation equilibrium constant were determined for ICAP
than for gal or lac sequences. Recently, it has been
suggested that the geometry of the CRP–DNA com-
plex plays a major role in the molecular mechanism of
gene transcription activation [37]. The FRET studies
of CRP–DNA interactions [38] have shown that the
lacP1 promoter bends symmetrically upon binding to

Materials
All chemicals purchased were of the highest quality and
purity. Acrylamide, EDTA, cAMP, dithiothreitol, phenyl-
methanesulfonyl fluoride, and Tris were purchased from
Sigma. N-Iodoacetylaminoethyl-1-naphthylamine-5-sulfo-
nate (1,5-I-AEDANS) was obtained from Molecular Probes
(Eugene, OR). The nutrients for bacterial growth were from
Gibco BRL. KCl, NaCl, NaH
2
PO
4
, and KI were obtained
either from Fluka (Buchs, Switzerland) or from Riedel-de-
Hae
¨
n (Seelze GmbH, Germany). dNTP, Pwo DNA poly-
merase, HindIII and EcoRI endonucleases, and T4 DNA
ligase were from Roche Molecular (Mannheim, Germany).
The absorption coefficients of the cyclic nucleotide and
1,5-I-AEDANS probe were 14 650 m
)1
Æcm
)1
at 259 nm [39]
and 6000 m
)1
Æcm
)1
at 340 nm [40], respectively. All measure-
ments were performed in buffers prepared in water that was

DNA was stored at )20 °C in an experimental buffer.
Purification of proteins
The isolation and purification procedures of wild-type CRP
and CRPW85A mutant were carried out in the same man-
ner as previously described [7]. The purity of the proteins
was confirmed by SDS ⁄ PAGE stained with Coomassie
blue. The concentration of proteins was determined by
absorption spectroscopy using the molar extinction coeffi-
cients 40 800 m
)1
Æcm
)1
[41] and 33 100 m
)1
Æcm
)1
at 278 nm
for CRPwt and CRPW85A [42] dimers, respectively.
The measurements were performed in 50 mm Tris ⁄ HCl
buffer, pH 7.9, containing 100 mm KCl and 1 mm EDTA
(buffer A), and 50 mm Tris ⁄ HCl buffer, pH 8.0, supple-
mented with 100 mm KCl and 1 mm EDTA (buffer B).
M. Tworzydło et al. CRP conformational changes induced by DNA and cAMP
FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS 1113
Fluorescence labeling of CRP
The preparation details of 1,5-I-AEDANS-labeled CRP
were as previously described [7].
The labeled proteins were purified on a Sephadex G-25
column equilibrated with buffer B. Fractions displaying an
absorbance at both 280 and 340 nm were combined and

F
0
¼
X
i
f
i
ð1 þ K
i
½QÞ expðV
i
½QÞ
ð3Þ
where F
0
and F are fluorescence intensities in the absence
and presence of quencher, Q, respectively, K
i
and V
i
are the
dynamic and static quenching constants, respectively, and f
i
is the fraction contribution (at the experimental excitation
and emission wavelengths) of component i. The quenching
rate constant k
q
was calculated as k
q
¼ K

ðkÞFðkÞð4Þ
In order to obtain the spectrum of each component, the
fluorescence spectra were collected at each quencher con-
centration, and a Stern–Volmer curve (Eqn 1) was fitted to
each set of data obtained at a given emission wavelength. A
simultaneous analysis of a series of fluorescence quenching
data enabled us to resolve the spectrum into components
according to (Eqn 2). The quenching data were fitted to the
Stern–Volmer equation according to a one-component or a
two-component model. The fits were characterized by the
minimum of the reduced v
2
and by the residual distribution
of the experimental data. In all calculations, the Stern–
Volmer constant, K
i
, as well as fraction f
i
were floating
parameters.
Lifetime measurements
Time-resolved intensity decay data were obtained by using
a K2 ISS phase ⁄ modulation frequency-domain fluorimeter,
equipped with a 300 W xenon lamp as a light source. The
excitation wavelength was set at 295 nm, with bandwidth
equal to 8 nm, by using a monochromator. Emission was
observed through a cut-off filter at a wavelength of
320 nm. All measurements were performed in buffer A at
20 °C. The light was modulated over the frequency range
from 10 to 200 MHz by using a Pockels cell modulator.

i
¼ a
i
s
i
⁄Sa
i
s
i
. The analysis of fluorescence
data was performed by using discrete exponential compo-
nents. The entire software for the data analysis was from
ISS. At an excitation wavelength, the protein sample had
an absorption of < 0.1. In each case the best-fit parameters
were obtained by minimization of the reduced v
2
value.
Stopped-flow FRET measurements
The stopped-flow fluorescence experiments were performed
on a SX-17 MV stopped-flow spectrophotometer obtained
from Applied Photophysics (Leatherhead, UK) in a two
syringe mode. The dead time of mixing was determined to
be less than 2 ms. The temperature in the stopped-flow unit
was maintained at 20 (± 0.1) °C using circulating water
CRP conformational changes induced by DNA and cAMP M. Tworzydło et al.
1114 FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS
from a thermostatically controlled bath. All measurements
were performed in buffer B.
The FRET kinetic experiments were used for quantitative
measurements of CRP–DNA binding in the presence of

Acknowledgements
This work was supported by grant no. 3 P04A 006 24
from the Ministry of Science and Informatics.
References
1 Busby S & Ebright R (1999) Transcription activation by
Catabolite Activator Protein (CAP). J Mol Biol 293,
199–213.
2 Harman JG (2001) Allosteric regulation of the cAMP
receptor protein. Biochim Biophys Acta 1547, 1–17.
3 Passner JM & Steitz TA (1997) The structure of a
CAP–DNA complex having two cAMP molecules
bound to each monomer. Proc Natl Acad Sci USA 94,
2843–2847.
4 de Crombrugghe B, Busby S & Buc H (1984) Cyclic
AMP receptor protein: role in transcription activation.
Science 224, 831–838.
5 Taniguchi T, O’Neill M & de Crombrugghe B (1979)
Interaction site of Escherichia coli cyclic AMP receptor
protein on DNA of galactose operon promoters. Proc
Natl Acad Sci USA 76, 5090–5094.
6 Passner JM, Schultz SC & Steitz TA (2000) Modeling
the cAMP-induced allosteric transition using the crystal
structure of CAP-cAMP at 2.1 A
˚
resolution. J Mol Biol
304, 847–859.
7 Polit A, Błaszczyk U & Wasylewski Z (2003) Steady-
state and time-resolved fluorescence studies of confor-
mational changes induced by cyclic AMP and DNA
binding to cyclic AMP receptor protein from Escheri-

19
F-n.m.r. studies of ligand binding to 5-fluorotrypto-
phan- and 3-fluorotyrosine-containing cyclic AMP
receptor protein from Escherichia coli. Biochem J 266,
545–552.
15 Tan G, Kelly P, Kim J & Wartell RM (1991) Compari-
son of cAMP receptor protein and a cAMP-independent
form of CRP by Raman spectroscopy and DNA bind-
ing. Biochemistry 30, 5076–5080.
16 Leu SF, Baker CH, Lee EJ & Harman JG (1999) Posi-
tion 127 amino acid substitutions affect the formation
of CRP:cAMP:lacP complexes but not
CRP:cAMP:RNA polymerase complexes at lacP. Bio-
chemistry 38, 6222–6230.
17 Małecki J, Polit A & Wasylewski Z (2000) Kinetic stu-
dies of cAMP-induced allosteric changes in cyclic AMP
receptor protein from Escherichia coli. J Biol Chem 275,
8480–8486.
18 Polit A, Bonarek P, Kepys B, Kedracka-Krok S,
Go
´
recki A & Wasylewski Z (2003) Kinetic studies of
M. Tworzydło et al. CRP conformational changes induced by DNA and cAMP
FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS 1115
cAMP-induced allosteric changes in T127I, S128A and
T127I ⁄ S128A mutants of cAMP receptor protein from
Escherichia coli. J Biol Chem 278, 43020–43026.
19 Błaszczyk U, Polit A, Guz A & Wasylewski Z (2002)
Interaction of cAMP receptor protein from Escherichia
coli with cAMP and DNA studied by dynamic light

gaard-Andersen L, Mironov AS, Pedersen H, Sukh-
odelets VV & Valentin-Hansen P (1991) Single amino
acid substitutions in the cAMP receptor protein specifi-
cally abolish regulation by the CytR repressor in
Escherichia coli. Proc Natl Acad Sci USA 88, 4921–
4925.
29 Chen Y & Barkley MD (1998) Toward understanding
tryptophan fluorescence in proteins. Biochemistry 37,
9976–9982.
30 Krueger S, Gregurick S, Shi Y, Wang S, Wladkowski
BD & Schwarz FP (2003) Entropic nature of the inter-
action between promoter bound CRP mutants and
RNA polymerase. Biochemistry 42, 1958–1968.
31 Baichoo N & Heyduk T (1998) DNA-induced confor-
mational changes in cyclic AMP receptor protein: detec-
tion and mapping by a protein footprinting technique
using multiple chemical proteases. J Mol Biol 290,
37–48.
32 Perez-Howard GM, Weil AP & Beechem JM (1995)
Yeast TATA binding protein interaction with DNA:
fluorescence determination of oligomeric state, equili-
brium binding, on-rate, and dissociation kinetics. Bio-
chemistry 34, 8005–8017.
33 Kwon H, Park S, Lee S, Lee D-K & Yang C-H (2001)
Determination of binding constant of transcription fac-
tor AP-1 and DNA. Application of inhibitors. Eur J
Biochem 268, 565–572.
34 Saecker RM, Tsodikov OV, McQuade KL, Schlax PE
Jr, Capp MW & Record MT Jr (2002) Kinetic studies
and structural models of the association of E. coli r70

42 Gill SC & von Hippel PH (1989) Calculation of protein
coefficients from amino acids sequence data. Anal Bio-
chem 182, 319–326.
43 Eilen E & Krakow JS (1977) Cyclic AMP-mediated
intersubunit disulfide crosslinking of the cyclic AMP
receptor protein of Escherichia coli. J Mol Biol 114,
47–60.
44 Schultz SC, Shields GC & Steitz TA (1991) Crystal
structure of a CAP–DNA complex: The DNA is bent
by 90 degrees. Science 253, 1001–1007.
CRP conformational changes induced by DNA and cAMP M. Tworzydło et al.
1116 FEBS Journal 272 (2005) 1103–1116 ª 2005 FEBS