Measurement of fluorescence energy transfer FRET between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP–cAMP2complex caused a substan-tial increase in FRET efficiency..
Trang 1Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP
Agnieszka Polit, Urszula Błaszczyk and Zygmunt Wasylewski
Department of Physical Biochemistry, Faculty of Biotechnology, Jagiellonian University, Krako´w, Poland
cAMP receptor protein (CRP), allosterically activated by
cAMP, regulates the expression of several genes in
Escheri-chia coli As binding of cAMP leads to undefined
conform-ational changes in CRP, we performed a steady-state and
time-resolved fluorescence study to show how the binding of
the ligand influences the structure and dynamics of the
protein We used CRP mutants containing a single
trypto-phan residue at position 85 or 13, and fluorescently labeled
with 1,5-I-AEDANS attached to Cys178 Binding of cAMP
in the CRP–(cAMP)2complex leads to changes in the Trp13
microenvironment, whereas its binding in the CRP–
(cAMP)4complex alters the surroundings of Trp85
Time-resolved anisotropy measurements indicated that cAMP
binding in the CRP–(cAMP)2complex led to a substantial
increase in the rotational mobility of the Trp13 residue
Measurement of fluorescence energy transfer (FRET)
between labeled Cys178 and Trp85 showed that the binding
of cAMP in the CRP–(cAMP)2complex caused a substan-tial increase in FRET efficiency This indicates a decrease in the distance between the two domains of the protein from 26.6 A˚ in apo-CRP to 18.7 A˚ in the CRP–(cAMP)2 com-plex The binding of cAMP in the CRP–(cAMP)4complex resulted in only a very small increase in FRET efficiency The average distance between the two domains in CRP–DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to 20 A˚ The spectral changes observed provide new structural information about the cAMP-induced allosteric activation of the protein
Keywords: allosteric regulation; cAMP receptor protein; emission anisotropy; Escherichia coli; fluorescence
cAMP receptor protein (CRP), which is allosterically
activated by cAMP, regulates transcription of over 100
genes in Escherichia coli [1,2] Upon binding the cyclic
nucleotide, CRP undergoes an allosteric conformational
change that allows it to bind specific DNA sequences with
increased affinity [3] CRP is a dimeric protein, composed of
two identical 209-amino-acid subunits Each subunit of
CRP has a molecular mass of 23.6 kDa, as deduced from
the amino-acid sequence Individual subunits fold into two
domains [4] The larger N-terminal domain (residues 1–133)
is responsible for dimerization of CRP and for interaction
with the allosteric effector, cAMP The smaller C-terminal
domain (residues 139–209) is responsible for interaction
with DNA through a helix–turn–helix motif CRP
recog-nizes a 22-bp, symmetric DNA site [5] Amino-acid residues
134–138 form a flexible hinge which covalently couples two domains Recent studies of the crystal structure of the CRP– DNA complex showed that each protein subunit binds two cAMP molecules with different affinities [6] Higher-affinity sites, where the nucleotide binds in the anti conformation, are buried within the N-terminal domains, whereas lower-affinity binding sites (where the bound cAMP has a syn conformation) are located at the interface formed by the two C-terminal domains of the CRP subunits, interacting with a helix–turn–helix motif and, indirectly, with the DNA Crystallographic observations have been supported by recent NMR [7] and isothermal titration calorimetry studies [8] Therefore, it has been suggested that CRP exists in three conformational states: free CRP, CRP with two cAMP molecules bound to N-terminal domains [CRP–(cAMP)2], and CRP with four cAMP molecules bound to both N-terminal and C-terminal domains [CRP–(cAMP)4] An earlier hypothesis suggested [9] that the three conforma-tional states of CRP consisted of the following species: free CRP, CRP–(cAMP)1and CRP–(cAMP)2, which has been reinterpreted by Passner & Steitz [6] It is important to note that the behavior of CRP at different concentrations of cAMP is essentially biphasic, so two different conformers exist at lower and higher concentrations of cAMP In the presence of 100 lMcAMP, CRP becomes activated and is able to recognize and bind specific DNA sequences and stimulate transcription [10], whereas at millimolar concen-trations of cAMP, there is a loss of affinity and sequence specificity for DNA binding and, consequently, loss of transcription stimulation [11] In the crystal phase, the CRP
Correspondence to Z Wasylewski, Department of Physical
Biochemistry, Faculty of Biotechnology, Jagiellonian University,
ul Gronostajowa 7, 30-387 Krako´w, Poland.
Fax: + 48 12 25 26 902, Tel.: + 48 12 25 26 122,
E-mail: wasylewski@mol.uj.edu.pl
Abbreviations: 1,5-I-AEDANS,
N-iodoacetylaminoethyl-1-naphthyl-amine-5-sulfonate; AEDANS-CRP, CRP covalently labeled with
1,5-I-AEDANS attached to Cys178; apo-CRP, unligated CRP;
CRP, cAMP receptor protein; FRET, fluorescence resonance
energy transfer.
(Received 31 October 2002, revised 19 December 2002,
accepted 3 February 2003)
Trang 2dimer with two molecules of cAMP bound to anti-cAMP
binding sites is asymmetric, i.e one monomer is in an open
form, in which the a-helices are swung out away from the
N-terminal domain, and the other monomer is in a closed
form, in which the a-helices are swung in close to the
N-terminal domain [4] X-ray crystal structure studies
revealed that the cAMP-ligated CRP dimer complexed to
a 30-bp DNA sequence is exclusively in the closed form [12]
Each subunit of CRP contains two tryptophan residues at
positions 13 and 85 (Fig 1) Both residues are located in the
N-terminal domain, Trp85 near the cAMP-binding pocket
and Trp13 on the surface of the protein Trp13 is much more
accessible to solvent than Trp85 19F-NMR studies have
revealed that binding of cAMP induces not only changes in
the immediate environment of the cAMP-binding site but
also far-reaching conformational changes (perturbation of
chemical shift of Trp13) [13] Fluorescence studies have
shown that Trp13 is responsible for 80% of the tryptophan
fluorescence in CRP with 20% of the signal originating
from Trp85 [14] Acrylamide-mediated and iodide-mediated
fluorescence quenching studies indicate that Trp13 is
solvent-exposed and accessible to the quenching agents On the other
hand, Trp85 is inaccessible to quenching agents Heyduk &
Lee [9] have shown that, at micromolar cAMP
concentra-tions, there is no detectable change in fluorescence intensity
of protein tryptophan residues The increase was only
detected in the millimolar range of cAMP concentration
Biochemical and biophysical studies have demonstrated
that binding of cAMP allosterically induces CRP to assume a
conformation that binds to DNA and interacts with RNA
polymerase However, the details of the mechanism by which
cAMP mediates the allosteric activation of CRP remain obscure, because the crystal structure of apo-CRP has not yet been elucidated Therefore, we decided to study the changes
in the apo-CRP structure induced by binding of cAMP and DNA by measuring fluorescence resonance energy transfer (FRET) and fluorescence anisotropy decay Two tryptophan residues, Trp13 and Trp85, were used as intrinsic donors for FRET analysis The fluorescence anisotropy decay was used
to investigate the dynamics of CRP
Experimental procedures
Materials Acrylamide, KCl, EDTA, phenylmethanesulfonyl fluoride, Tris and N-iodoacetylaminoethyl-1-naphthylamine-5-sulfo-nate (1,5-I-AEDANS) were purchased from Sigma cAMP and dithiothreitol were from Fluka The Fractogel EMD
SO3650 (M) was from Merck, and Q-Sepharose Fast Flow, Sephacryl S-200 HR and Sephadex G-25 were from Amersham Pharmacia Biotech DNA sequence containing the CRP-binding sites was from TIB MOLBIOL (Poznan´, Poland) The nutrients for bacterial growth were from Life Technologies All other chemicals were analytical-grade products of POCh-Gliwice (Gliwice, Poland) All measure-ments were performed in buffers prepared in water purified with the Millipore system
The sequences of duplexes used in this study were as follows:
lac(26 bp), 5¢-ATTAATGTGAGTTAGCTCACTCATT A-3¢ and 3¢-TAATTACACTCAATCGAGTGAGTAAT-5¢; gal (26 bp), 5¢-AAAAGTGTGACATGGAATAAATT AGT-3¢ and 3¢-TTTTCACACTGTACCTTATTTAATCA-5¢; ICAP (28 bp), 5¢-AATTAATGTGACATATGTCACAT TAATT-3¢ and 3¢-TTAATTACACTGTATACAGTGTAAT TAA-5¢
The recognition half-sites are shown in bold An equimolar amount of complementary strand was added, and the mixture was heated for 1 min at 96C and slowly cooled to room temperature The double-stranded DNA was stored at)20 C in experimental buffer
Protein purification The tryptophans at positions 13 and 85 of CRP were replaced with phenylalanine and alanine, respectively The mutagen-esis was performed using the overlap extension method with PwoDNA polymerase pHA7 plasmid encoding mutant crp genes was introduced into E coli strain M182Dcrp, kindly provided by Dr S Busby (The University of Birmingham, UK) The bacteria were grown on Luria–Bertani medium at
37C overnight in a Biostat B fermentor from B Braun Biotech International (Melsungen, Germany) Proteins were purified at 4C, essentially as described previously [15] but with one modification After ion-exchange chromatography
on Q-Sepharose, the proteins were additionally purified by gel filtration on Sephacryl S-200 HR After this procedure, the proteins were highly pure (> 97%), as judged by SDS/ PAGE and Coomassie Brilliant Blue staining
For spectrophotometric determination of concentrations, the following absorption coefficients were used: 14 650
)1Æcm)1at 259 nm for cAMP [16] and 6000 )1Æcm)1at
Fig 1 Structure of the CRP dimer The locations of tryptophan
resi-dues are marked in red, and the location of Cys178 residue is indicated
in yellow The figure was generated with WEBLAB VIEWERPRO (version
3.7) using atomic coordinates for the CRP–cAMP complex [29] The
coordinates were obtained from the Brookhaven Protein Data Bank
(accession code 1G6N).
Trang 3340 nm for AEDANS [18] Absorption coefficients of CRP
mutants were determined using the method described
elsewhere [19] as 29 700M )1Æcm)1and 33 100M )1Æcm)1at
278 nm for the W13F and W85A dimers, respectively
Measurements were performed in 50 mM Tris/HCl
buffer, pH 8.0, containing 100 mM KCl and 1 mM
EDTA (buffer A), and 50 mM Tris/HCl buffer, pH 7.8,
supplemented with 100 mM KCl and 1 mM EDTA
(buffer B)
Fluorescence labeling of CRP
Covalent modification of Trp mutants with 1,5-I-AEDANS
was carried out as described elsewhere [20] with several
modifications The protein and label were mixed at a molar
ratio of 1 : 10 and incubated at room temperature for 2 h,
and then at 4C overnight in the dark The labeled CRP
was purified on a Sephadex G-25 column equilibrated with
buffer A Fractions displaying a high absorbance at both
280 and 340 nm were combined and dialyzed extensively
against buffer A
Mapping of modified residues
Mapping of labeled residues was performed as described
previously [21] with several modifications Peptides were
separated by an HPLC system consisting of (a) a Shimadzu
LC-9A pump equipped with FCV-9AL low-pressure
pro-portioning valve, (b) a Knauer A0263 manual injector
equipped with a 100-lL loop, (c) a Supelcosil LC-318
HPLC (5 lm) cartridge column (250· 4.6 mm) with
20· 2.1 mm Supelguard LC-318 precolumn, (d) a
Merck-Hitachi L-4000A detector, (e) a Shimadzu RF-535
fluores-cence monitor, and (f) a Shimadzu Class-VP 1-2 hardware/
software system for data acquisition and analysis Solvent A
was 0.1% trifluoroacetic acid in water, and solvent B was
0.08% trifluoroacetic acid in 80% acetonitrile A linear
gradient of 10–70% solvent B over 40 min was applied at a
flow rate of 1 mLÆmin)1, with spectrophotometric detection
at 215 nm and fluorescence detection at an excitation
wavelength of 336 nm and an emission wavelength of
490 nm
Steady-state fluorescence measurements
Steady-state fluorescence was measured with an Hitachi
F-4500 spectrofluorimeter All studies were carried out at
room temperature and excitation at 295 nm The
experi-ments were conducted in buffer A or buffer B The protein
solution had an initial absorbance at the excitation
wave-length lower than 0.1
The effect of cAMP on tryptophan fluorescence was
monitored by a fluorescence titration of CRPW13F and
CRPW85A Tryptophan emission was scanned from 310 to
480 nm When energy transfer was measured, the emission
spectra were recorded in the range 310–570 nm The
fluorescence quantum yield of the donor in the absence of
the acceptor (QD) was calculated from the equation:
QD¼ QRF
SDARF
SRFAD
ð1Þ
where SD and SRF are the respective areas under the emission spectra of the donor and a reference compound, and ARF and AD are the respective absorbances of the reference compound and donor at the excitation wave-length QRFis the quantum yield of the reference compound
L-tryptophan and was taken to be 0.14 in water at 25C [22]
All spectra were corrected for sample dilution and the inner filter effect, introduced by cAMP and DNA at the excitation wavelength, according to the following formula [23]
Fcor¼ F 10ðPþDAÞ=2 ð2Þ where F and Fcorare fluorescence intensity before and after the correction, and P and DA denote the initial sample absorbance at the excitation wavelength and the change in absorbance introduced by the ligand, respectively
Time-resolved fluorescence measurements Fluorescence decays were measured using a homemade time-correlated single-photon counting system based on Ortec electronics (Oak Ridge, USA) It consisted of (a) a Philips 2020Q photomultiplier with a 1.5-ns response time, (b) a 1-GHz preamplifier, (c) a quad constant fraction discriminator model 935, and (d) a time-to-amplitude converter (TAC) model 457 A nanosecond flash lamp nF
900 from Edinburgh Instruments was used as a light source (e) In the case of anisotropy measurements, (f) Glan– Thompson prism polarizers were also used
All measurements were performed at 20 ± 0.2C Before measurements, all samples were filtered through a microporous filter (0.45 lm; Millipore) to remove insoluble impurities
FRET measurements Energy transfer was observed between the tryptophan residues and the 1,5-I-AEDANS moiety covalently attached to Cys178 The tryptophans were excited at 297 nm Fluorescence decays were observed
at wavelengths between 320 and 400 nm using two cut-off filters Measurements were performed in buffer A Fluor-escence decays were recorded at a resolution of 23 ps per channel, resulting in a total time window of 100 ns Intensity decay data were analyzed using the following multiexponential decay law:
It¼X
i
aiexpðt=siÞ ð3Þ
where aiand siare the pre-exponential factor and decay time of component i, respectively The fractional fluores-cence intensity of each component is defined as ƒi¼ aisi/
Saisi The data were analyzed with the software from Edinburgh Instruments Best-fit parameters were obtained
by minimization of the reduced v2value
The average efficiency of energy transferÆEæ was calcu-lated from the average donor lifetime in the presenceÆsDAæ and absence of acceptorÆsDæ
< E >¼ 1 <sDA>
<sD> ð4Þ
Trang 4The average lifetime was obtained from the equation:
<s >¼
P
i
ais2 i
P
i
AsÆsDAæ and ÆsDæ are obtained without the need to know the
absolute protein concentrations, uncertainties associated
with protein concentration determination are eliminated in
the time-resolved fluorescence measurements
The average distance between the donor–acceptor pair
ÆRæ was calculated from the equation:
< R >¼ R0hE1 11=6i
½ ˚AA ð6Þ where R0 is the Fo¨rster critical distance (the distance at
which 50% energy transfer occurs) R0is given by:
R0¼ 9:78 103j2n4QDJð Þk1=6
½ ˚AA ð7Þ where n is the refractive index of the medium, QDis the
quantum yield of the donor, J(k) is a spectral overlap
integral of the donor fluorescence and acceptor absorption,
and j2 is the orientation factor and accounts for relative
orientation of the donor emission and acceptor absorption
transition dipole Generally, j2is assumed to be equal to
2/3, which is the value for donors and acceptors that
randomized by rotational diffusion before energy transfer
Fluorescence anisotropy decay measurements The W13F
and W85A CRP mutants were used to measure the
rotational correlation time of the protein The excitation
wavelength for tryptophan residues was 297 nm
Fluores-cence anisotropy decays were observed using a cut-off
filter > 320 nm Experiments were performed at several
concentrations of the proteins (1.0–8.5 lM) for each species
The sample was excited with vertically polarized light
Fluorescence anisotropy decays with vertical and horizontal
emission polarization were alternatively recorded All
measurements were repeated at least twice for each sample
Fluorescence anisotropy decays were recorded at a
resolu-tion of 46 ps per channel, resulting in a total time window
of 200 ns
Anisotropy decay data were analyzed according to the
impulse reconvolution model decay law:
R tð Þ ¼ R1þXn
i¼1
Aiexpðt=hiÞ ð8Þ
where Ai are the amplitudes of the components with
rotational correlation time hI, and Rlis limiting anisotropy
The time-zero anisotropy r(0) was obtained from the
equation:
rð Þ ¼ R0 1þXn
i¼1
In each case, the best-fit parameters were obtained by
minimization of the reduced v2 test value The v2 and
residuals distribution were utilized to judge the goodness of
the fit The software used for analysis was from Edinburgh
Instruments
Results
Fluorescence labeling of CRP Each subunit of CRP possesses three cysteine residues, two
in the N-terminal domain (Cys19 and Cys92) and one in the C-terminal domain (Cys178) Only Cys178 can be chemi-cally modified under native conditions; Cys19 and Cys92 seem to be buried [24,25] To confirm the selectivity of the labeling, CRP mutants modified with 1,5-IAEDANS were denaturated and completely digested with trypsin and chymotrypsin The peptides liberated were examined by HPLC As expected, only one peptide fragment had been modified with thiol-reactive probe Thus, we conclude that CRP was uniformly labeled with 1,5-IAEDANS at the SH group of Cys178
The stoichiometry of the labeling was determined from the absorption spectrum of the labeled CRP When CRP was incubated with the fluorescence reagent, 1,5-I-AEDANS, at pH 8.0, a mean of 2 mol was bound per mol protein dimer The effect of the label on the secondary structure of CRP was investigated using CD spectroscopy No differences were observed between the modified and unmodified variants of CRP (data not shown) The insertion of the fluorescent probes also did not significantly alter the biological activity of CRP [20]
Steady-state fluorescence data The effect of cAMP on tryptophan fluorescence was monitored Changes in CRP tryptophan fluorescence were monitored by titrating the CRP solution with
1–2-lL aliquots of concentrated cAMP solution Measure-ments were performed in the cAMP concentration range
50 lM to 1 mM The fluorescence emission spectra of Trp13 and Trp85 in the presence and absence of cAMP are given in Fig 2A and Fig 3A, respectively When CRP was titrated with cAMP, the fluorescence intensity
of the Trp13 residue decreased with increasing ligand concentration However, this decrease could only by detected in the micromolar range of cAMP concentra-tions In addition, the emission maximum of Trp13 shifted from 342.5 nm to 340 nm The reduction in fluorescence intensity and the blue shift indicate a conformational transition of CRPW85A on binding of cAMP The effect of different concentrations of cAMP
on the fluorescence intensity of Trp13 is shown in Fig 2B When 200 lMcAMP was added to the solution
of CRPW85A, a 13% decrease in fluorescence intensity was observed
In contrast with the observation made with CRPW85A, the addition of cAMP to CRPW13F caused an increase in the fluorescence intensity of Trp85 with no change in the emission maximum The maximum wavelength of emission for CRPW13F was 339 nm The effect of different concentrations of cAMP on the fluorescence intensity of Trp85 is shown in Fig 3B In the case of Trp85, the addition
of 200 lM cAMP caused only a very small change in the fluorescence intensity, increasing it by 3.4% A pro-nounced increase in the Trp85 fluorescence intensity was
Trang 5detected only at high concentrations of cAMP (> 2 mM)
(data not shown)
Typical fluorescence spectra of CRPW13F, unmodified
and modified with 1,5-I-AEDANS, are shown in Fig 4
When excited at 295 nm, tryptophan residues in the
unlabeled protein had a fluorescence emission maximum
near 339 nm In the presence of 1,5-I-AEDANS,
trypto-phan fluorescence intensity was significantly reduced
com-pared with an approximately equal concentration of an
unmodified protein The maximum wavelength of
trypto-phan emission in the labeled mutant W13F was shifted to
327 nm The addition of cAMP and DNA increased
energy transfer from Trp85 residue to the AEDANS
moiety
The quantum yields of tryptophan fluorescence at
25C in buffer A were determined to be 0.09 for mutant
CRPW13F alone and 0.094 for mutant CRPW13F in the
presence of 200 lM cAMP The quantum yield of the donor increased upon protein–DNA complex formation The change in the observed value of the quantum yield was 20% A similar result was obtained for CRP– (cAMP)4
Time-resolved fluorescence data FRET measurements Lifetime measurements on the mutant CRPW13F labeled with 1,5-I-AEDANS indicated
a decreased lifetime of the tryptophan fluorescence, as expected when energy transfer occurs Figure 5 shows the time-dependent donor decays for the proteins bearing donor alone and those with donor and acceptor In the case of mutant CRPW85A, no energy transfer was observed Lifetime measurements were repeated several times for each species The fluorescence decays were
Fig 3 Fluorescence emission spectra of
CRPW13F in the absence (—) and presence of
cAMP at 50 l M (ÆÆÆÆ) and 1 m M cAMP (- - -).
Excitation was at 295 nm All spectra were
recorded in buffer B, pH 7.8 The inset in the
plot shows fluorescence intensity change in
Trp85 as a function of cAMP concentration.
F and F 0 are the fluorescence intensities of the
protein in the presence and absence of the
ligand, respectively The range of cAMP
con-centrations used was from 50 l M to 1 m M
The line was drawn only to indicate the trend
of the data.
Fig 2 Fluorescence emission spectra of
CRPW85A in the absence (—) and presence of
cAMP at 50 l M (ÆÆÆÆ) and 1 m M cAMP (- - -).
Excitation was at 295 nm All spectra were
recorded in buffer B, pH 7.8 The inset shows
fluorescence intensity change in Trp13 as a
function of cAMP concentration F and F 0 are
the fluorescence intensities of the protein in the
presence and absence of the ligand,
respect-ively The range of cAMP concentrations used
was from 50 l M to 1 m M The line was drawn
only to indicate the trend of the data.
Trang 6analyzed as a multiexponential decay, and for each case the
double exponential decay was characterized by lower values
of reduced v2 In some cases the triple exponential decays
were recorded; however, the pre-exponential factor a3for
these fits was close to zero, and therefore this component of
the decay is not included in the calculated average values of
fluorescence lifetimesÆsDæ and ÆsDAæ The values for average
lifetimes of Trp85 in the absence and presence of acceptor
and efficiency of energy transfer are presented in Table 1
The transfer efficiency varied between apoprotein and after
binding cAMP at micromolar or millimolar concentrations
In the absence of a specific ligand, CRPW13F showed an
efficiency of transfer of 24.2 ± 8.1% The addition of
cAMP had a significant effect on energy transfer The value
for CRPW13F with two cAMP molecules bound to
anti-cAMP-binding sites was considerably higher
(72.3 ± 2.5%) than for apoprotein The result determined
for the mutant W13F in the presence of 2 mMcAMP was
similar to the above value, averaging 74.3 ± 2.4% Energy
transfer in CRPW13F bound to the specific fragments of
DNA was also measured and displayed similar values of
efficiency for each complex examined ( 62%)
The energy-transfer efficiency values were used to
calcu-late the average distance between the donor and the
acceptor To approximate the distance between the
Trp85–Cys178 pair, we assumed that the Fo¨rster distance
for the tryptophan–IAEDANS pair was 22 A˚ [26,27] The
estimated distances are shown in Table 1 There was a
significant difference between the calculated distance in
apo-CRPW13F and CRPW13F complexed with cAMP
and DNA
Time-domain anisotropy data The fluorescence
aniso-tropy decays of Trp13 and Trp85 were measured to
determine the changes in the rotational diffusion of CRP
after cAMP binding The anisotropy decay of Trp13 in the
CRP complexed with two cAMP molecules is shown in
Fig 6A Analysis of the anisotropy decays was carried out
according to Eqn (8) with an increasing number of
exponents, until the fit no longer improved In all cases,
the anisotropy decays of Trp13 and Trp85 could be described by one exponent with single rotational correlation time Addition of the second exponent did not significantly alter the goodness of fit The v2 value obtained with the single-exponential and double-exponential analysis indi-cates that the two-component analysis is not significantly better than the one-component analysis Also the distribu-tion of the residuals did not improve on the addidistribu-tion of the second component (Fig 6B,C) However, the data obtained for the single exponential analysis suggest the presence of an additional segmental mobility This is evident from appar-ent time-zero anisotropy r(0), which is lower than the fundamental anisotropy r0of tryptophan at this excitation wavelength [28] For both tryptophan residues, the initial anisotropy was in the range 0.22–0.23 (Table 2) It indicates that anisotropy decay contains a fast component which cannot be resolved with our device
The anisotropy decay parameters for various CRP species are reported in Table 2 The mean ± SD value
of rotational correlation times determined from the study
of fluorescence anisotropy decays of Trp85 in the absence
of specific ligand was 20.5 ± 2.4 ns The value for Trp13 was considerably lower, averaging 15.3 ± 1.8 ns The addition of 100 lM cAMP probably did not affect the rotational correlation time of CRPW13F The uncer-tainty of this value was too large to ascertain any changes in the CRP dynamic on cAMP binding Trp13 exhibited different behavior The rotational correlation time determined from the study of fluorescence aniso-tropy decays of Trp13 in the presence of cAMP decreased to 10.45 ± 3.0 ns
Discussion
cAMP binding to CRP has been studied using a variety of methods, which have shown that the ligand binding mediates changes in the protein conformation It is believed that these changes allow the protein molecule to switch from the low-affinity and nonspecific DNA-binding state to the state characterized by high affinity and sequence specificity
Fig 4 Fluorescence emission spectra of unmodified and modified CRPW13F The excitation wavelength was 295 nm and emission was scanned from 310 to 570 nm Measurements were performed at 25 C in buffer A, pH 8.0 (— Æ Æ —) Unmodified CRPW13F; (—) modified CRPW13F; ( -) modified CRPW13F in the presence of 200 l M
cAMP; (ÆÆÆÆ) modified CRPW13F bound to DNA in the presence of cAMP.
Trang 7for the DNA promoter [2] As the X-ray crystal structure of
apo-CRP has not yet been resolved, it is believed that the
binding of cAMP, which leads to a switch to active protein
conformation, involves subunit realignment and hinge
reorientation between the protein domains [2,29] For a
long time, it has been a paradigm that CRP undergoes a
cAMP concentration-dependent transition between three
conformations: apo-CRP, CRP–(cAMP)1 and CRP–
(cAMP), and each conformer possesses a unique structure
and activity [2] The reinterpretation of this paradigm was proposed by Passner & Steitz [6] on the basis of the crystal structure of the CRP–cAMP complex, and it has recently been supported by NMR [7] and isothermal titration calorimetry [8] studies in solution NMR experiments have shown that CRP possesses two anti-cAMP-binding sites in each monomer, and the next two syn-cAMP sites are formed by an allosteric conformational change in the protein on biding of two anti-AMP at the N-terminal
Fig 5 Trp85 fluorescence intensity decays for mutant CRPW13F without and with 1,5-I-AEDANS covalently attached to Cys178 The dark grey dotted curve shows the intensity decay of the donor alone (D), and the grey dotted curve shows the intensity decay of the donor in the presence of the acceptor (DA) The black solid lines and weighted residuals (lower panels) are for the best triple exponential fits Experiments were performed in buffer A at 20 C.
Trang 8domain [7] The isothermal titration calorimetry
measure-ments demonstrated that, at low cAMP concentration,
there are two identical interactive high-affinity sites for
cAMP and at least one low-affinity cAMP-binding site at
high concentration of the ligand [8]
The idea of the four cAMP-binding sites in CRP, for its
anticonformation (at low concentration of the ligand) and
the next two binding it in syn conformation (at high cAMP
concentration) has been used to describe the results of fast
kinetic studies [15] and investigations with dynamic light
scattering and time-resolved fluorescence anisotropy
meas-urements [30] In these studies, we have shown that the
binding of cAMP in anti conformation in the N-terminal
domain of CRP leads to the conformational changes in the
helix–turn–helix motif of the C-terminal domain,
respon-sible for the interaction with DNA, as well as to the changes
in the global hydrodynamic structure of CRP The
satura-tion of the low-affinity sites with cAMP in syn conformasatura-tion
of cAMP results in the changes in the microenvironment of
the Trp85 residue, localized in the N-terminal domain of the
protein, without further substantial changes in the global
hydrodynamic structure [30]
The results presented in this report provide further
evidence for conformational changes induced by cAMP
binding to the anti-cAMP-binding sites of CRP, which in
turn trigger specific pathways of signal transmission from
the cyclic nucleotide-binding domain to the DNA-binding
domain of the protein We have used single
tryptophan-containing mutants of CRP The mutations were localized
in the N-terminal domain at position 85 or 13 in order to
follow conformational changes in their microenvironment
on binding of cAMP, both in anti-and syn-conformation
We have also used AEDANS for fluorescent labeling of
Cys178, located at the turn of the helix–turn–helix motif, in
order to detect FRET between Trp85 and the label We
have shown that binding of cAMP at a concentration of
200 lM to anti-cAMP-binding sites results in 13%
decrease in the Trp13 fluorescence intensity along with the
blue shift in its maximum of emission by 2.5 nm Probable
candidates for quenching residues in CRP are Thr10,
Asn109 and His17, which are located within a distance up to
5 A˚, as has been determined from the X-ray crystal
structure of the CRP–cAMP complex (PDB code 1G6N)
[29] The observed changes in the microenvironment of
Trp13 on filling of the high-affinity sites are also supported
by the time-resolved anisotropy measurements Binding of
the ligand to anti-cAMP-binding sites results in a decrease in
rotational correlation time by 5 ns, from the value of
15.3 ns, detected for apo-CRP, to the value of 10.4 ns for
CRP–(cAMP) complex The decrease in rotational time
indicates an increase in the mobility of helix A of the protein As Trp13 is located in the vicinity of the activation region AR2 of the protein [1], which is responsible for the activation of the second class of E coli promoters such as galP1, one can speculate that this conformational change may play an important role in a signal transmission in the protein molecule, which in turn may allow CRP to adopt a conformation appropriate for the interaction with the aNTD domain of RNA polymerase in the transcription complex On the other hand, Trp13 of CRP directly interacts with another gene regulatory protein, CytR [31], and the observed conformational changes in Trp13 micro-environments on cAMP binding to anti-cAMP sites may play a significant role in the CRP–CytR–DNA complex
In contrast with Trp13 of CRP, binding of cAMP to anti-cAMP-binding sites does not lead to a significant change in fluorescence intensity of Trp85, and only a 3.4% increase
in the intensity has been observed at 200 lM cAMP However, cAMP binding to the syn-cAMP-binding sites at concentration of the ligand of 1 mMcauses a 6% increase
in its fluorescence intensity, which indicates that this residue
is sensitive to cAMP binding to low-affinity sites The rotational correlation time of Trp85 in apo-CRP of 20.5 ns indicates that this residue is immobilized within the N-terminal domain of the protein and exhibits motion characteristic of the whole protein (within experimental error), while the respective value for the AEDANS-labeled apo-CRP has been estimated at 23.3 ns [30] Binding of cAMP to anti-cAMP-binding sites increases the rotational correlation time to 22 ns for the CRP–(cAMP)2complex, which is much lower than the correlation time of 30 ns determined for this complex using CRP labeled at Cys178 with the AEDANS fluorescent probe [30] These discre-pancies can probably be explained by the fact that the average fluorescence lifetime of Trp85, 5.6 ns in the case
of the CRP–(cAMP)2 complex, is too short to allow observations of the longer rotational correlation times The allosteric activation of CRP involves conformational changes in the N-terminal domain of the protein and leads
to changes in the CRP molecule, enabling it to recognize the specific DNA sequence [1,2] As the crystal structure of apo-CRP has not yet been established, it was suggested that cAMP binding may cause reorientation of the coiled-coil C helices, consequently altering the relative position of the protein dimer subunits [29] These authors also suggested that, in the absence of cAMP in apo-CRP, some b strands
of the N-terminal domain of the protein may collapse into the cAMP-binding pocket, causing reorientation of the smaller domain in relation to the larger one, and bringing these domains closer together This suggestion has been
Table 1 Summary of energy transfer measurements In the CRPW13F–(cAMP) 2 complex, the concentration of cAMP was 200 l M , whereas in the case of CRPW13F–(cAMP) 4 the concentration of cAMP was 2 m M The molar ratio CRP to DNA in the protein–DNA complex was 1 : 1.
Trang 9supported recently by NMR studies [7]; these authors argue that binding in solution of two cAMP molecules to high-affinity anti-cAMP-binding sites at the N-terminal domain causes the C-terminal domain to shift further to the N-terminal domain of CRP To confirm this suggestion,
we used FRET to detect the distance between the C-terminal and N-terminal domains of CRP on binding of cAMP in anti
as well as syn conformation to the protein For this purpose,
we used time-resolved fluorescence lifetime measurements
Table 2 Parameters of Trp13 and Trp85 anisotropy decays in the
presence and absence of cAMP.
CRPW13F–(cAMP) 2 22.1 ± 6.9 0.23 ± 0.03 1.036
CRPW85A–(cAMP) 2 10.45 ± 3.0 0.23 ± 0.03 1.146
Fig 6 Time-domain fluorescence anisotropy decay of Trp13 in the presence of 100 l M cAMP The solid line corresponds to the best single exponential fit of the data (dotted curve) according to Eqn (8) The grey cross-haired curve represents the lamp profile The plots of the residuals for the best single exponential fit (B) and the double exponential fit (C) are also shown Measurements were performed at 20 C in buffer B, pH 8.0, with a CRPW85A concentration of 1.1 l M Excitation was at 297 nm.
Trang 10using single tryptophan-containing mutants of CRP
Fluo-rescence energy transfer could be detected between Trp85,
localized close to the cAMP-binding pocket of the
N-terminal domain, and Cys178, fluorescently labeled by
AEDANS, localized in the helix–turn–helix motif of the
C-terminal domain of the protein The lifetimes obtained for
the fluorescence donor, Trp85, indicate that binding of
cAMP to anti-cAMP-binding sites leads to a dramatic
increase in FRET efficiency This observation clearly shows a
decrease in the average distance between the two domains of
CRP on cAMP binding If one assumes the Fo¨rster distance,
R0, for the pair donor–acceptor such as tryptophan–
AEDANS to be 22 A˚ [26,27], the distance between Trp85
and Cys178-AEDANS in the apo-CRP can be calculated to
be 26.6 A˚ This distance decreases by about 8 A˚ to 18.7 A˚ on
binding of cAMP to the anti-cAMP-binding sites of the
protein The distance between the sulfur atom of Cys178 and
the C9–C10 bond of the indole ring of Trp85, derived from
the crystal structure of CRP–(cAMP)2(PDB code 1G6N) is
18.9 A˚ and 21.9 A˚ for the subunit present in the closed and
open conformation, respectively [29] The structural
asym-metry of the CRP–(cAMP)2 complex resulting from
con-formational differences between subunits has been
questioned [32], and from molecular dynamics simulation,
it has been predicted that, in solution, both subunits of CRP
adopt a closed conformation If this is so, the distance (equal
to 18.7 A˚), determined in this work by FRET, is in good
agreement with the value of 18.9 A˚ predicted for the closed
conformation This supports experimentally the dynamic
simulation studies [32] and indicates that, in solution, both of
the protein subunits exist in closed conformation in the
CRP–(cAMP)2complex
Because a variety of spectroscopic effects, at least in
theory, could influence energy transfer efficiency, one can
argue that the good agreement determined for the distance
between Cys178 and Trp85 residues in CRP–(cAMP)2may
also result from the assumed value of Fo¨rster distance R0
However, as binding of cAMP at a concentration of 200 lM
to CRP leads only to a 4.3% increase in the fluorescence
quantum yields from the value of 0.09 for apo-CRP to the
value of 0.094 for the CRP–(cAMP)2 complex, and no
substantial changes in the shape of the emission spectra of
the donor have been observed, this justifies the lack of the
alteration of the Fo¨rster distance between apo and holo
forms of the protein As the AEDANS label attached to the
Cys178 enjoys local freedom of movement, in both
apo-CRP and the apo-CRP–(cAMP)2 complex [30], the distance
obtained from the crystal structure between the sulfur atom
of Cys178 and the indole ring of Trp85 seems to be realistic
We have also tried to measure fluorescence energy transfer
between Trp13 and Cys178-AEDANS; however, we have
not detected any energy transfer in either the apo-or
holo-form This could be because of the distance between the two
residues, which is about 45 A˚, as can be calculated from the
crystal structure of CRP–(cAMP)2[29]
Binding of cAMP in syn conformation to the
low-affinity binding sites in the CRP–(cAMP)4 complex leads
to only a small increase in the efficiency of energy
transfer, which, with an assumed R0 value of 22 A˚,
corresponds to the small decrease in average distance
between the N-terminal and C-terminal domains of CRP,
estimated at 18.4 A˚ However, the fluorescence quantum
yield of the Trp85 donor increases by 20% at a concentration of cAMP of 2 mM from the value characteristic of apo-CRP, which in turn may be responsible for this very small change We have also measured the distance between the two domains of CRP
in the complexes with DNA containing various
sequenc-es, such as lac and gal promoters and with the symmetric sequence ICAP For each CRP–DNA complex, the increase in the distance between the two CRP domains has been observed with the average distance of 20.2 A˚ This value is in good agreement with the value of 20.7 A˚, calculated from the crystal structure of the CRP– DNA complex [33]
The present results show that the binding of anti-cAMP
in the CRP–(cAMP)2complex results in the movement of the C-terminal domain of CRP by 8 A˚ towards the N-terminal domain, which in consequence leads to rearrangement of DNA-binding domains and cAMP-binding domains of the protein This finding clarifies the suggestion derived from the NMR measurements [7] that the C-terminus is closer to the N-terminal domain in apo-CRP than in cAMP-bound apo-CRP Binding of cAMP to anti-cAMP-binding sites leads to an increase in the structural dynamic motion around Trp13, which is close
to the activation region AR2, responsible for the interac-tion of CRP with the a subunit of RNA polymerase The changes in the CRP dynamics on cAMP binding have recently been observed by the hydrogen exchange method [34] In that paper, it was shown that binding of the ligand to the protein causes the C-terminal domain of CRP to become more flexible, in contrast with the N-terminal domain which is shifted to a less dynamic conformation Our results extend this observation and suggest that the binding of cAMP to anti-cAMP-binding sites of CRP leads to the increase in the structural dynamic motion of at least Trp13, which is located in the N-terminal domain of the protein
Acknowledgements
We are grateful to Dr S Garges for supplying us with the plasmid for production of CRP This work was supported by grant no.
6 P04A 031 16 from the State Committee for Scientific Research.
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