Báo cáo khoa học: Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli ppt

We applied the iodide and acrylamide fluorescence quenching method in order to study how different DNA sequences and cAMP binding induce the conformational changes in the CRP molecule.. W

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

interactions; fluorescence quenching; FRET,

fast kinetics

Correspondence

Z Wasylewski, Department of Physical

Biochemistry, Faculty of Biotechnology,

Jagiellonian University, ul Gronostajowa 7,

30–387 Krako´w, Poland

Fax: +48 12 66 46 902

Tel: +48 12 66 46 122

E-mail: wasylewski@mol.uj.edu.pl

(Received 29 July 2004, revised 22

November 2004, accepted 21 December

2004)

doi:10.1111/j.1742-4658.2005.04540.x

Cyclic AMP receptor protein (CRP) regulates the expression of more then

100 genes in Escherichia coli It is known that the allosteric activation of CRP by cAMP involves a long-distance signal transmission from the N-ter-minal cAMP-binding domain to the C-terN-ter-minal domain of CRP responsible for the interactions with specific sequences of DNA In this report we have used a CRP mutant containing a single Trp13 located in the N-terminal domain of the protein We applied the iodide and acrylamide fluorescence quenching method in order to study how different DNA sequences and cAMP binding induce the conformational changes in the CRP molecule The results presented provide evidence for the occurrence of a long-distance conformational signal transduction within the protein from the C-terminal DNA-binding domain to the N-terminal domain of CRP This conformational signal transmission depends on the promoter sequence We also used the stopped-flow and Fo¨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.

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

impossible However, it has been suggested from the

crystal structure studies that the cAMP-induced

allo-steric transition may involve a change in relative

ori-entation of the subunits and a change in oriori-entation

of the DNA-binding domain relative to the

cAMP-binding domain [6] Indeed, our Fo¨rster resonance

energy transfer (FRET) measurements show that the

binding of anticAMP in the CRP–(cAMP)2 complex

results in a movement of the C-terminal domain of

CRP by
8 A˚ towards the N-terminal domain [7]

As in the CRP–(cAMP)2 complex the anticAMP is

buried within the N-terminal domain of the protein

located at least 10 A˚ away from the hinge region,

the allosteric activation of CRP must involve a

long-distance signal transmission within the protein Recent

studies [8] suggest that this long-distance

communica-tion between the two CRP domains and subunits

involves the Asp138 residue, located in the CRP hinge

region, which represents part of the signal

transduc-tion network

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 signal transmission from the C-terminal domain to the N-terminal domain of the protein Furthermore, we present the kinetics of DNA–CRP interactions, as determined by using FRET measurements, between labeled Cys178 of CRP and fluorescently labeled DNA sequences (Fig 1)

The mechanism of the cAMP-induced long-distance structural communication within the CRP remains an important part of our understanding of the mechan-ism underlying the transcription-regulating activity

of this protein However, it is an open question as to how the binding of the CRP–(cAMP)2 complex to different specific promoter DNA sequences can trigger the conformational changes in the protein that may consequently lead to changes in the interactions between the activator and other participants of the transcription machinery Does it involve a conforma-tional signal transmission from the C-terminal domain

of CRP through the hinge region to the N-terminal domain? We believe that elucidation of the signal transduction pathway from the different DNA sequences to the activation regions in CRP may pro-vide a structural paradigm for understanding the tran-scription activation process Therefore, we suggest that the CRP does not act by the simple ‘recruitment’ mechanism in transcription machinery, as has been suggested recently [12], but behaves as a very dynamic entity

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).

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 squench-ingle 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, KSV The fluorescence quenching data were

analyzed according to Eqn (3), by using a nonlinear

least-squares procedure The analysis was conducted

for all the quenching data, i.e for about 40 different

emission spectra Judging by the calculated v2 value

and the residual distribution, the phenomenon can be

described by a two-component model in which one

component in the protein is more available for the

quencher and characterized by KSV1¼ 9.61 m)1 and

an f1 of
0.55, while the other component is less

accessible to the iodide with KSV2¼ 1.69 m)1 and

f2¼ 0.45 The best theoretical-fit line calculated for

the given emission wavelength is shown in Fig 2A

Similar results were obtained for the CRPW85A–

(cAMP)2 complex The Stern–Volmer plot also curved

down (data not shown) The binding of cAMP resulted

in a small increase of the KSV1value from 9.61 m)1 to

10.08 m)1 and the more visible increase of the KSV2

value from 1.69 m)1to 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, KSV¼ 6.62 m)1, and the values of

a static quenching constant, V, are 0.84 m)1 and

0.27 m)1, respectively The fitting parameters for iodide

and acrylamide quenching are given in Table 1

Figure 3 shows, for the first time, the spectra of

CRP, containing a single Trp13 residue, resolved into

components by using the

fluorescence-quenching-resolved spectra (FQRS) method, using iodide as a

quencher The component characterized by a higher

Stern–Volmer constant (9.61 m)1) was found to exhibit

a maximum at 350 nm and to account for 55% of the fluorescence emission The second component, charac-terized by the average KSV¼ 1.69 m)1, is responsible for
45% of the total emission and has a maximum

at 338 nm

Fig 2 (A) Typical Stern–Volmer plot for iodide quenching of CRPW85A ( ) The solid line represents the best fit with the fol-lowing parameters: KSV1¼ 9.11 M )1, f

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,

KSV¼ 5.64 M )1, V¼ 0.74 M )1, f¼ 1; CRPW85A–(cAMP) 2 , KSV¼ 6.45 M )1, V¼ 0.22 M )1, f¼ 1 The excitation was at 295 nm and the emission at 340 nm.

FQRS spectra of CRPW85A with cAMP are

repre-sented in Fig 4 The binding of the ligand results in a

blue shift of the total spectrum maximum from about

342 nm to 340 nm The more quenchable component

exhibits a kmax at 344 nm, whereas the maximum of

the less quenchable component remains unchanged at

338 nm The maxima of the resolved spectra and their

relative intensities, measured as the areas under each

of the resolved spectra, are given in Table 1

Analogous measurements were performed for CRP–

DNA complexes Figure 5A,B shows typical Stern–

Volmer plots obtained for iodide and acrylamide

quenching of the CRPW85A mutant bound to ICAP,

lacand gal sequences

For all three DNA fragments, the Stern–Volmer

plots of fluorescence quenching by iodide exhibit a

downward curvature, and the best fits were obtained

with a two-component model in which one component

is quenchable and the second remains inaccessible for

the quencher In order to prove that the downward

curvature was not a result of the ionic strength

chan-ges when iodide was added, the titration of the

CRPW85A–DNA complexes with KCl was performed

and it did not lead to any substantial changes in

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 KSV1 is 5.54 m)1, and for

CRPW85A–gal, the value of KSV2 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 V¼ 1.35 m)1 The KSV

for the CRPW85A–gal complex was lower than for the complexes with the ICAP and lac sequences and equaled 5.53 m)1

The total fluorescence emission of all three CRPW85A–DNA complexes had maxima at the same wavelength as the CRPW85A–(cAMP)2 complex (Figs 6, 7 and 8), i.e at 340 nm The resolved spectra which correspond to the unquenchable components have maxima at around 338 nm, while the maxima of quenchable components are located at around 344 nm The detailed parameters of the resolved spectra of the CRPW85A–DNA complexes, with iodide used as a quencher, are presented in Table 1

Time-resolved fluorescence data Fluorescence lifetime measurements of the CRPW85A mutant and its complexes with cAMP and DNA were conducted using an excitation wavelength equal to

295 nm Phase and modulation were analyzed by using single- and double-exponential decay models The bet-ter fits, i.e of lower values of the reduced v2, were obtained for a double-exponential model The values

of mean fluorescence lifetimes, defined as sm¼ Sfis, are presented in Table 2

Table 1 Fluorescence quenching parameters for CRPW85A, CRPW85A–(cAMP)2and 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 KSVand 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.

SV2 ( M )1) V (M)1) f

1

FQRS

k maks1 (nm) k maks2 (nm) Iodide quenching

Acrylamide quenching

Kinetics of DNA binding to CRP

A FRET has been used to study the kinetics of CRP–

DNA interactions The fluorescence characteristics of

CRP-conjugated IAEDANS, with an excitation at

340 nm and a maximum emission at 480 nm, suggest

that it can be used as a donor fluorophore

Oligonucleo-tides covalently labeled with fluorescein were used as

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 (kobs) are linear (Fig 9) and the values of koff and the association-rate parameter, kon, listed in Table 3, were determined as the intercept and the slope that are valid for a single-step bimolecular association:

Fig 4 Fluorescence-quenching-resolved spectra (FQRS) of CRPW85A–(cAMP)2with excitation at 295 nm Iodide was used as

a quencher The upper panel represents a plot of Stern–Volmer constants as a function of the emission wavelength The lower panel shows the FQRS: ( ) the total emission spectrum with a maximum at about 340 nm; (h) the more quenchable component with a maximum at
344 nm, characterized by an average value of

KSV1¼ 10.08 M )1and a fraction f

1 ¼ 0.57; ( ) the less quenchable component with the maximum at
338 nm, characterized by an average value of K SV2 ¼ 2.85 M )1and a fraction f

2 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

KSV1¼ 9.61 M )1and 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 )1and a fraction f

2 ¼ 0.45.

CRPðcAMPÞ2þ DNA !kon

koff DNACRPðcAMPÞ2 and which

kobs¼ koffþ kon½CRPAEDANS ð1Þ

with the total concentration used of IAEDANS attached to CRP denoted as [CRP–AEDANS] An equilibrium binding constant can be calculated from the ratio of the rate constants konand koffas follows:

Ka¼kon

koff

ð2Þ

Association constants (Ka) 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, KSV1¼ 4.63 M )1, f

1 ¼ 0.81 (B) Typical Stern–Volmer plots for acrylamide quenching of CRPW85A

com-plexes with DNA The solid lines represent the best fits with the

following parameters: (e) CRPW85A–ICAP, KSV¼ 5.92 M )1, f¼ 1;

(d) CRPW85A–lac, K SV ¼ 5.74 M )1, f¼ 1; (.) CRPW85A–gal,

KSV¼ 5.30 M )1, V¼ 1.16 M )1, f¼ 1 The excitation was at 295 nm

and the emission at 340 nm.

Fig 6 Fluorescence-quenching-resolved spectra (FQRS) of CRPW85A–ICAP 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: (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 f1¼ 0.80; and ( ) the unquenchable component with

a maximum at
338 nm, characterized by an average value of

K SV2 ¼ 0.00 M )1and a fraction f

2 ¼ 0.20.

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˚ in apo–

CRP to 18.7 A˚ in the CRP–(cAMP)2 complex [7] The

details of the structural mechanism of CRP activation

by a cAMP have not been established because of the

lack of an X-ray structure for apo–CRP However, it

may be expected that the binding of an allosteric

inductor, cAMP, as well as an interaction of the

protein with the specific DNA promoter sequences in

solution can lead to changes in the protein activation regions, which in turn allows CRP to interact with the

a subunit of RNA polymerase Recently [7] we have suggested that cAMP binding to anti sites leads to an increase in the structural dynamic motion around the Trp13 residue, which is close to the activation region AR2, responsible for the interaction of CRP with RNA polymerase [10]

The tryptophan residue is widely used as an intrinsic fluorescence probe to observe changes in protein struc-ture [20] High indole sensitivity to its microenviron-ment in a protein moiety can be used to follow protein structural changes, especially if the complicated emis-sion of tryptophan residues may be resolved into com-ponents The difficulties in the interpretation of its fluorescence emission result from the dynamics of pro-tein structure and the multiple ground-state conformers, each of which is characterized by distinct tryptophan

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

KSV1¼ 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 )1and 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 ¼ 5.02 M )1and a fraction f

1 ¼ 0.81; and ( ) the unquenchable com-ponent with a maximum at about 337 nm, characterized by an aver-age value of K SV2 ¼ 0.00 M )1and a fraction f

2 ¼ 0.19.

residue microenvironments [20] To resolve the fluores-cence emission spectra into components in a protein containing multiple tryptophan residues, advanced techniques for analyzing fluorescence decay emission may be used [20] Under steady-state conditions, the quenching processes may be analyzed by the external quenchers by using the FQRS method [21,22] Quench-ing experiments are especially useful in studyQuench-ing the changes in the conformation of proteins that may be induced by ligand binding If the studied protein pos-sesses several tryptophan residues, then the interpret-ation of a change in the quenchability is more difficult However, site-directed mutagenesis may be used to 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)2and CRPW85A–DNA com-plexes Experiments were performed at 20
C in Tris buffer, pH 7.9 In the experiments with CRPW85A complexed to cAMP and DNA, the concentration of cAMP was 100 l M Excitation was at 295 nm and emission through a cut-off filter The error did not exceed 5%.

Species

s 1

s 2

(ns)

s m

(ns)

Iodide quenching Acrylamide quenching

k q1 ( M )1Æs)1)

x 10)1

k q2 ( M )1Æs)1)

x 10)1

k q1 ( M )1Æs)1)

x 10)1

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

and emission > 500 nm.

Table 3 Kinetic and thermodynamic parameters describing the

binding of lacF, galF and ICAPF to the wild-type cyclic AMP

recep-tor protein (CRPwt) The values are derived from experiments

con-ducted at 20
C, in 50 m M Tris ⁄ HCl buffer, containing 100 m M KCl,

1 m M EDTA, pH 8.0, in the presence of 200 l M cAMP Kinetic and

thermodynamic parameters are defined as detailed in the

Experi-mental procedures The error is the SD of fitted parameters.

Complex k off (s)1) k on (s)1Æ M )1)· 10 6 K a ( M )1)· 10 5

CRPwt–ICAPF 5.8 ± 0.6 3.4 ± 0.2 5.9 ± 0.9

CRPwt–lacF 8.5 ± 0.9 1.1 ± 0.2 1.2 ± 0.3

CRPwt–galF 5.1 ± 0.9 2.4 ± 0.2 4.7 ± 0.9

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,

KSV¼ 9.6 m)1 If the average lifetime of Trp13 is

assumed to be 2.3 ns, then the bimolecular

rate-quenching constant, kq, can be calculated as

4.16· 109m)1Æs)1 This value is typical of the

trypto-phan residues in proteins exposed to a solvent [25]

The second conformational state of CRP can be

char-acterized by a relatively bluer emission with the

maxi-mum at 338 nm In this conformational state of CRP,

the Trp13 residue is much less accessible to the iodide

quencher, as can be judged by a bimolecular rate

quenching constant, kq¼ 0.73 · 109m)1Æs)1 These

two conformational states of CRP are not

distinguish-able by acrylamide (another quencher used in this

study) The acrylamide bimolecular rate quenching

constant, kq, equaling 2.49· 109m)1Æs)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 usquench-ing 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 decay of the Trp13 residue is more complex than that described by a double-exponential decay, but we have had little success in trying to resolve the fluorescence

to more components on our apparatus As a result, when we calculated the bimolecular rate quenching constants, kq, we obtained values of the average Trp13 lifetime instead of the values of lifetimes of the resolved components

Binding of cAMP to anticAMP-binding sites leads

to significant changes in the fluorescing properties of Trp13 of CRP–(cAMP)2, including changes in the maximum fluorescence emission of the component more quenchanable by iodide, as well as the increase

in bimolecular rate-quenching constants, kq, for iodide and acrylamide (Tables 1 and 2) These results provide further evidence for changes in the protein dynamics induced by cAMP binding to the anti sites of CRP in the CRP–(cAMP)2 complex, in the surroundings of Trp13 As the distance between the Trp13 residue and the anticAMP molecule, both located in the N-terminal domain in the CRP–(cAMP)2 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 conformaactiva-tional 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

CRP–(cAMP)2 complex after binding specific DNA,

which occurs from the C-terminal domain to the

N-terminal domain of the protein We have shown this

by using the Trp13-containing mutant of CRP as well

as the iodide and acrylamide fluorescence quenching

method in order to follow the influence of DNA

bind-ing on the conformational changes in its

microenviron-ment We have used various DNA sequences: lac, gal

and ICAP The synthetic artificial ICAP DNA

posses-ses a symmetrical sequence, which binds with high

affinity to the CRP HTH motifs, and the lac and gal

DNA sequences represent the CRP-binding sites from

class I and class II CRP-dependent promoters,

respect-ively [1] Our iodide fluorescence-quenching

measure-ments of DNA–CRP complexes show that CRP still

exists in two different conformational states, but they

significantly differ in Trp13 microenvironments which

determine the Trp13 fluorescing properties These

dif-ferences do not result from an increase in the ionic

strength of the solution upon titration of the sample

by KI, because the titration performed with KCl up to

a concentration similar to that of KI did not cause

any change in fluorescence of the complexes (data not

shown) The best fits for all the tested DNA sequences,

as judged by reduced v2 values as well as residual

dis-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 kq 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

in solution involves a further conformational signal

transduction from the C-terminal domain to the

N-ter-minal domain of CRP, and the magnitude of this

conformational transduction solely depends on the promoter DNA sequence responsible for this inter-action This suggestion is in agreement with small angle neutron scattering measurements of the CRP– DNA complex, which indicate that this structural change in the N-terminal domain of the protein occurs upon binding of DNA to the C-terminal domain of CRP [30] Our fluorescence studies of CRP–DNA interactions presented here also agree with the results

of Baichoo & Heyduk [31], which were obtained by protein footprinting techniques These authors, using chemical proteases of different charge, size and hydro-phobicity, suggested that the binding of DNA in solu-tion induces conformasolu-tional changes in the N-terminal domain of CRP, close to the activating region, AR2 Our fast-kinetics study presented here has also shown that the DNA–CRP interactions depend on the sequence of the 26 bp DNA fragments The bimole-cular rate constant values of 3.4· 106m)1Æs)1, 1.1· 106m)1Æs)1 and 2.4· 106m)1Æs)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)3and 10)2s)1 that has been found for other proteins which interact with DNA [32–34] The observed differences in the dis-sociation rate constants may result from the fast association of CRP with DNA, which leads to forma-tion of the low-affinity CRP–DNA complex This is followed by the slow process of conformational chan-ges in the C-terminal domains of CRP, which permit formation of the high-affinity complex As the kinetics

of CRP–DNA interactions have been detected by determining the resonance energy transfer between fluorescently labeled CRP and DNA, we have been able to observe only the first step of the association process without detecting any possible consecutive reactions However, we have observed the fluorescence intensity changes of CRP–AEDANS upon the binding

of DNA sequences, which result from the conforma-tional changes in the C-terminal domain of the pro-tein The values of CRP–DNA association equilibrium constants, Ka, calculated from the rate constants pre-sented in Table 3, are equal to 5.9· 105m)1, 1.2· 105m)1and 4.7· 105m)1for ICAP, lac and gal, respectively These values are slightly lower than the association constants of 4.0· 105m)1 and 11.1·

105m)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