From the Center for Advanced Research in Biotechnology/National
Institute of Standards and Technology, Rockville, Maryland 20850 and the
National Institute of Standards and Technology
Center for Neutron Research, National Institute of Standards and
Technology, Gaithersburg, Maryland 20899
Small angle neutron scattering (SANS)
measurements were performed on solutions of cAMP receptor protein (CRP)
and on solutions of the T127L,S128A double mutant of CRP (CRP*) in
D2O K3PO4 buffer containing
0.5 M KCl, in the absence and presence of 3',5' cyclic adenosine monophosphate (cAMP). Energy-minimized structures of the CRP
were calculated by minimization of the x-ray crystallographic structure
of CRP in either the exclusively "closed" form where the
-helices of the carboxyl-terminal domain are folded close to the
amino-terminal domain and in the exclusively "open" form where the
-helices of the carboxyl-terminal domain are folded away from the
amino-terminal domain. Neutron scattering models show that the CRP SANS
data follow closely the data curve predicted for unligated CRP in the
open form, whereas the cAMP-ligated data are more in agreement with the
data predicted for the minimized cAMP-ligated CRP structure in the
closed form. Thus, it appears that CRP undergoes a conformational
change from the open form to the closed form in solution upon ligation
with cAMP. The SANS data from the CRP* and cAMP-ligated CRP* are
coincidental, which implies that there is very little structural
difference between the two species of CRP*. This is in agreement with
in vivo results, which show that whereas CRP activates
transcription in the cell only in the presence of cAMP, CRP* activates
transcription in the absence of cAMP, implying that CRP* is already in
the correct conformation for the activation of transcription.
 |
INTRODUCTION |
The binding of 3',5' cyclic adenosine monophosphate (cAMP) to cAMP
receptor protein (CRP)1
activates the transcription of over 20 genes that code for the catabolite enzymes involved in carbohydrate metabolism in
Escherichia coli. Results from NMR (1, 2), Raman
spectroscopy (3), proteolysis studies (4), small angle x-ray scattering
measurements (5), and isothermal titration calorimetry (6) indicate
that upon binding to cAMP, CRP undergoes a conformational change to a
conformation that promotes specific binding to the catabolite operons
to activate transcription in the presence of RNA polymerase. The nature
of this conformational change is not known, because only the structures
of the cAMP-ligated CRP complex and of the DNA-cAMP-ligated CRP complex
have been determined from x-ray crystallography studies (7, 8).
CRP exists as a homodimer of 45,000 g mol
1 consisting of
a
-pleated sheet amino-terminal domain, which contains the cAMP
binding site and a carboxyl-terminal domain consisting of several
-helices that bind to specific DNA sequences. In the crystal phase,
the cAMP-ligated CRP dimer is asymmetric where one monomer is in an "open" form in which the
-helices are swung out away from the amino-terminal domain and the other monomer is in a "closed" form in which the
-helices are swung in close to the amino-terminal domain (7). The amino acid sequence connecting the two domains is,
thus, called the "hinge" region. The x-ray crystal structure of the
cAMP-ligated CRP dimer complexed with a 30-base pair DNA sequence is
exclusively in the closed form (8). Recent energy minimization
computations of the two forms in solution show that the lowest energy
conformation of the cAMP-ligated CRP dimer is exclusively the closed
form in solution (9). The implication is that the asymmetry of the CRP
dimer in the crystal lattice is due to crystal packing forces and that
the structural change responsible for the activation of transcription
is a change from the open to the closed form in solution. This is
substantiated by NMR measurements on fluorinated derivatives of CRP,
which imply that the conformational change involves the hinge region
(1), and proton NMR measurements, which show that CRP in solution
tightens up and becomes more rigid upon binding of cAMP (2).
There is, however, evidence that the conformational change may not
simply involve a transition from the open to the closed form of CRP
upon binding to cAMP in solution. A comparison of the secondary
structure of free CRP and cAMP-ligated CRP by Raman spectroscopy (3)
shows a structural shift from 44%
-helix, 28%
-strand, 18%
turn, and 10% undefined in the free form to 37%
-helix, 33%
-strand, 17% turn, and 12% undefined in the ligated form, implying
that the conformational change does not conserve the
-helical and
-strand structure as it would in a shift of the CRP dimer from the
exclusively open to the closed form in solution. A conformational
change involving alterations in the
-helical and
-strand
structure of CRP is also implied by an empirical model of CRP secondary
structure based on its sequence. This sequence-based empirical model
predicts
-helices in unligated CRP in place of the
-strands in
the amino-terminal domain of the x-ray crystallographic structure of
the cAMP-ligated CRP complex (10).
In addition to the contradictory evidence concerning the nature of the
conformational change in CRP, there is also experimental evidence that
a cAMP-induced conformational change is not always necessary for the
activation of transcription. For example, in vivo
transcription was observed to be activated even in the absence of cAMP
by a mutant of CRP where two amino acid residues have been replaced in
the amino-terminal domain of CRP, T127L and S128A (CRP*) (11). This
implies that CRP* is already in the transcriptionally activated
conformation and that cAMP binding is unlikely to initiate a
conformational change.
Small angle neutron scattering (SANS) measurements have been employed
to determine the conformation and size of proteins in solution (12).
Using D2O as the solvent to maximize the difference in
neutron scattering between the solvent and the protein, the conformation of the protein can be determined from the dependence of
the scattered neutron intensity on the scattering angle and the size of
the protein in terms of the radius of gyration
(Rg) can be determined from the scattered
neutron intensity near zero scattering angle (13). In addition, the
SANS data can be compared with scattered neutron intensity curves
generated from the energy-minimized crystallographic x-ray structure of
the protein to determine in more detail the conformation of the protein
in solution.
In this investigation, SANS measurements were performed on solutions of
CRP in the absence and presence of cAMP and compared with model
scattered intensity curves calculated from the energy-minimized x-ray
structures of CRP dimer in exclusively the closed and in exclusively
the open forms. For comparison, SANS measurements were also performed
on solutions of CRP* in the absence and presence of cAMP.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The characterization and mutagenesis of the wild
type and mutant of CRP have been described previously by Moore (11). A New Brunswick Fermentor Scientific, Inc. SF116 was also used in place
of the 5-liter shaker flasks for preparations of CRP and CRP*. An
Amersham Pharmacia Biotech Phast SDS-electrophoresis gel with 0.1 M Tris-HCl buffer (pH 7.0) containing 0.2 mass % bromphenol blue and 20 volume % glycerol was run to determine the
purity of the protein preparations. A protein analysis was also
performed by comparing the absorbance at 562 nm of samples of the
solution to those of bovine serum albumin solutions after adding 4 mass
% CuSO4 and bicinchoninic acid reagent from Pierce. The
protein solutions were dialyzed at 4 °C in 50 mM
potassium phosphate-potassium hydroxide buffer (pH = 7.0)
containing 0.2 mM sodium EDTA, 0.2 mM
dithiothreitol, 0.5 mM KCl, and 5 volume % glycerol
(phosphate buffer) in D2O with two changes in the buffer solution. The concentrations of the CRP and the mutant were determined by UV absorption spectroscopy using an extinction coefficient of
3.5 × 104 cm liter mol
1 at 280 nm (14).
The ligand solutions were made up by dissolving known masses of the
sodium salt of the cyclic nucleotide in the D2O buffer. The
sodium salt of cAMP, potassium phosphate, NaOH, EDTA, dithiothreitol,
glycerol, Tris, HCl, bromphenol blue, and KCl were reagent grade from
Sigma.
SANS Measurements--
The SANS measurements were performed on
the NG3 30 m SANS instrument at the National Institute of
Standards and Technology Center for Neutron Research in Gaithersburg,
MD (15). Unligated CRP solutions were measured at 0.036 and 0.096 mM and unligated CRP* at 0.049 and 0.138 mM in
the D2O solvent. CRP was also measured at both
concentrations in the presence of 0.270 and 17.1 mM cAMP, whereas CRP* solutions at both concentrations were measured in the
presence of 17.1 mM cAMP. To limit aggregation effects, the lower concentration solutions were used to obtain the data at the lower
scattering angles where information about the overall size of the
protein is obtained, whereas the higher concentration solutions were
used to obtain the higher angle data, which provide information about
the shape of the protein. In the higher angle scattering region,
aggregation effects are much smaller than in the lower angle region,
but the scattered intensity is also much lower, so a higher
concentration of CRP and CRP* in solution is needed in the higher angle
region.
Data were taken at a wavelength
= 5 Å, with a spread 
/
= 0.34 for the CRP samples and 
/
= 0.15 for the CRP* samples. A
sample-to-detector distance of 5.5 m and a source-to-sample distance of 7.02 m were used for the 0.036 and 0.049 mM samples to obtain the lower angle data. The 0.096 and
0.138 mM samples were measured at a sample-to-detector
distance of 1.5 m and a source-to-sample distance of 3.92 m
to obtain the higher angle data. The source and sample apertures were
5.0 and 1.27 cm, respectively. Neutrons were detected on a 64.0 × 64.0-cm two-dimensional position-sensitive detector with 1.0-cm
resolution. The center of the detector was offset by 20.0 cm at both
sample-to-detector distance positions to provide a large overlap
between the two configurations and to measure to a large enough
scattering angle to allow an accurate determination of the incoherent
background scattering due to hydrogen in the sample. The scattered
intensity, I, was corrected for solvent absorbance and
scattering on a pixel-by-pixel basis using the relation,
|
(Eq. 1)
|
where Tsample and
Tsolvent are the measured transmissions of
neutrons through the sample and solvent, respectively. The corrected data were placed on an absolute scale by calibration against the scattering from a silica gel standard sample and then radially averaged
to obtain I(Q) as a function of Q,
where Q = 4
sin
/
, and 2
is the scattering
angle.
The radially averaged data exhibit a distinct flat region beyond
Q = 0.35 Å
1 due to the incoherent
scattering from hydrogen in the samples. This background scattering was
subtracted from the data taken at both instrument configurations,
normalizing for the difference in the protein concentrations. The
background-subtracted data for both configurations were simultaneously
corrected (desmeared) for instrument resolution effects, and Fourier
transformed to obtain the distance distribution function,
P(r), using two different methods: the Svergun
method (16), as implemented in the program GNOM (17), and the Glatter
method (18), as implemented in the program GLATTER, with resolution
effects incorporated by Skov Pederson et al. (19). Both
methods assume the scattering results from a dilute, monodispersed
solution of globular particles. In addition, it is assumed that the
particles have a sharp boundary such that there is a maximum diameter,
Dmax, beyond which there is no significant
scattering mass. The desmeared scattered intensity, I(Q), is related to P(r) by
the equation,
|
(Eq. 2)
|
where the two conditions, P(0) = 0 and
P(2r
Dmax) = 0, are satisfied. 4
P(r) is defined as the number
of distances, r, between scattering centers within the
particle. The I(Q) versus Q
data taken at both instrument configurations are combined in the
desmearing process, and I(Q) versus
Q data were obtained over a usable range 0.012
Q
0.3 Å
1. The results from the two
desmearing methods were averaged to produce the final desmeared
I(Q) versus Q data and
P(r) versus r data.
In the small angle limit, the scattered intensity can be approximated
by the Guinier equation (13),
|
(Eq. 3)
|
where I(0) is the scattered intensity at
Q = 0. The radius of gyration of the particle,
Rg, is defined as the mean square distance from
the center of scattering mass. A plot of ln(I)
versus Q2 should contain a linear
region with slope Rg2/3 and
intercept ln(I(0)). Thus, Rg can be
obtained directly from the SANS measurements. The range of validity for
the Guinier approximation depends upon the shape of the particle.
However, Guinier fits are generally made in a Q range
centered about the relation, QRg ~ 1.0.
Details of the data desmearing procedure are illustrated in Fig.
1, which shows the smeared
I(Q) versus Q data for the
unligated CRP sample measured in D2O solvent. The
dotted line is the best fit to the data using the Glatter
(18, 19) method, in which the data are represented by a series of
B-splines. The data were fitted several times using different estimates
for Dmax. The fit that resulted in the smoothest
P(r) function for which the
Dmax value was clearly not too large, and for
which the calculated I(Q) still fitted well to
the experimental data, was chosen as the best solution.
Dmax values of 65 ± 5 Å in Equation 2
proved to fit the smeared data best in all cases. The "x" points
show the desmeared data resulting from the dotted line fit. Errors on
the desmeared data were determined by averaging the best-fit results
from both the Svergun (16, 17) and Glatter (18, 19) methods.

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Fig. 1.
SANS intensity versus Q
data from 0.036 and 0.096 mM unliganded CRP in
D2O solvent, uncorrected for instrument resolution effects
( ), along with the best fit obtained using the Glatter (18, 19)
desmearing method (· · · ·), and the resultant desmeared
data (×).
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The I(Q) versus Q data
obtained from SANS measurements can be compared with that calculated
from the energy-minimized x-ray crystallographic structure using a
Monte Carlo technique (20). Since hydrogen atoms are not included in
the x-ray structure, each amino acid is better represented by a
homogeneous sphere positioned according to its
-carbon coordinates.
The size and neutron scattering strength of each sphere are calculated
from the known volume and chemical composition of the amino acid,
respectively, thus taking into account the contribution of hydrogen.
Once a sphere has been drawn around each amino acid, a box is drawn
around the entire molecule, with the option to include a region of
bound water up to 5 Å thick. The bulk solvent can be any
H2O/D2O mixture, and the protein can be
hydrogenated or deuterated. Points are then generated at random within
the box. The distance distribution function,
P(r), for the total volume is calculated directly
in real space by making a histogram representing the distances between all possible pairs of points, weighted according to the product of the
neutron scattering strengths at each point. Rg
is calculated directly from the second moment of
P(r) and I(Q) is found
using Equation 2.
Energy Minimization Computations--
The x-ray crystallographic
structure of the cAMP-ligated CRP dimer shows that it can exist in an
open and closed form in the crystal phase. The CRP dimers were
generated from the Brookhaven Protein Data Bank file 3GAP.pdb
exclusively in either the closed or open form by superimposing a copy
of the monomer in the closed from on the monomer in the open form and
vice versa (20). The energetics of the structures were then minimized
using the steepest descent method for 1000 iterations, after which the
conjugate gradient method was used until convergence was achieved
(maximum derivative less than 0.04 kJ mol
1).
Computational results were obtained using software programs from
Biosym/MSI of San Diego. Energy minimizations, using the AMBER
forcefield, were done with the Discover program, and structure analysis
and comparison were performed with the DeCipher program. Energy-minimized structures for the unliganded CRP were generated in
the same manner, using the cAMP-ligated x-ray structure, with the cAMP
removed, as the starting structure.
 |
RESULTS |
The desmeared I(Q) versus
Q data for the CRP and the cAMP-ligated CRP solutions are
plotted in Fig. 2. It should be
emphasized that at the levels of 0.036 mM CRP and 0.270 mM cAMP concentrations, the CRP is only about 90% ligated,
so that the scattering would contain some contribution from unligated
CRP. At higher cAMP concentrations of approximately 17.1 mM, where the CRP would be fully ligated (21), aggregation
was observed in the CRP solutions as indicated by a sharp upward
inflection of the neutron scattering intensity near Q = 0 and, thus, these data were not analyzed. The scattering curve for CRP
in the presence of cAMP deviates significantly as shown in Fig. 2 from
that of the unligated CRP above Q = 0.15 Å
1. This deviation results from a conformational change
in CRP upon ligation with cAMP and is in contrast to the coincidence of
the desmeared SANS data from solutions of CRP* in the absence and presence of 17.1 mM cAMP shown in Fig.
3. (Aggregation at this cAMP
concentration was not observed for the CRP* solutions.) The nearly
coincidental curves in Fig. 3 show that CRP*, contrary to CRP,
undergoes very little conformational change in the presence of cAMP, as
implied by the in vivo activation of transcription by CRP*
in the absence of cAMP.

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Fig. 2.
Desmeared SANS intensity versus
Q data from unliganded CRP in D2O solvent
( ) and from CRP + 0.270 mM cAMP in D2O
solvent ( ), where the CRP is about 90% in the cAMP-ligated
form.
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Fig. 3.
Desmeared SANS intensity versus
Q from 0.049 and 0.138 mM unligated CRP*
( ) and CRP* + 17.1 mM cAMP ( ) in D2O
solvent.
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The radii of gyration calculated from the second moment of the distance
distribution function, P(r), using all of the
desmeared data in Figs. 2 and 3 are presented in Table
I. For comparison, Table I also lists the
radii of gyration obtained by averaging the results from several
Guinier fits over the Q range limited to 0.02
Q
0.08 Å
1 for unligated and ligated CRP
and CRP*. The radii of gyration calculated by the two methods are
essentially the same for the unligated and cAMP-ligated CRP* solutions
(approximately 22.3 ± 0.3 Å), indicating again virtually no
difference between the size of CRP* and cAMP-ligated CRP*. This is in
contrast with the results from the CRP solutions, using the two
methods, which exhibit a consistent shrinkage of about 1.0 Å upon cAMP
ligation. The slightly larger Rg values
determined for CRP from the Guinier fits compared with those determine
from the 2nd moment of P(r) may be due to the
effect of some aggregation in the sample on the Guinier-derived
Rg. The Rg values
calculated from P(r) are not significantly
affected by small deviations in the scattering curve due to aggregation
effects at small Q, where the Guinier-derived Rg values were determined.
The desmeared data in Fig. 2 are compared in Fig.
4 to model scattering curves calculated
from their corresponding energy-minimized x-ray structures, using 2000 randomly placed points in the volume occupied by the molecule. Three
iterations were performed for each of four structures, i.e.
unligated CRP dimer in the exclusively open and closed forms, and
cAMP-ligated CRP dimer in the exclusively open and closed forms. The
final model curves represent the average of the three iterations. The
unligated CRP scattered intensity, along with the model intensities
calculated from the energy-minimized x-ray structures of CRP with cAMP
removed, are plotted in Fig. 4A as a function of the
dimensionless parameter, Q·Rg
(QRg). Plotting the curves in this manner
maximizes the differences in structure due to changes in overall shape,
while minimizing changes in Rg. In Fig.
4A, the experimental desmeared SANS data fit very closely the model curve for unligated CRP exclusively in the open form. The
close agreement between the desmeared SANS data and the model curve of
CRP in the open form indicates that the open form of CRP is the
preferred conformation of unligated CRP in solution. This is further
substantiated by the radii of gyration obtained from the second moment
of P(r) for the energy-minimized x-ray structures
listed in Table I. The Rg value from the
desmeared unligated CRP data is 22.0 ± 0.2 Å, which compares
more favorably with 20.5 ± 0.4 Å for the minimized, exclusively
open form of CRP than with 18.7 ± 0.3 Å for the minimized,
exclusively closed form of CRP.

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Fig. 4.
Desmeared SANS intensity versus
QRg data from unligated CRP
(A) and cAMP-ligated CRP (B) in
D2O solvent ( ), along with model SANS curves
calculated from energy-minimized x-ray structures of unligated CRP
(A) and cAMP-ligated CRP (B) in the closed form
( ) and in the open form ( ).
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The desmeared scattered intensity for cAMP-ligated CRP is plotted in
Fig. 4B as a function of QRg, along
with the model intensities obtained from the energy-minimized x-ray
structures of cAMP-ligated CRP. The model curves for the minimized
exclusively closed and open forms of the cAMP-ligated CRP are less
separated than the corresponding model curves for unligated CRP in Fig.
4A and have Rg values of
approximately 19.2 ± 0.3 Å in each case, compared with
Rg = 21.6 ± 0.2 Å for the desmeared data
in Table I. However, the desmeared data appear to follow more closely
the model curve calculated from the minimized structure of cAMP-ligated
CRP dimer exclusively in the closed form.
The SANS results show that the conformational change that occurs in CRP
upon ligation with cAMP can well be described as a change from the open
to the more compact closed form of CRP. This is further evident in the
distance distribution functions, P(r), plotted
for unligated and cAMP-ligated CRP in Fig.
5A. In the presence of cAMP,
the peak shifts slightly toward smaller r values, indicating
that cAMP-ligated CRP is more compact than nonligated CRP. Fig.
5B shows the P(r) functions obtained
from the energy-minimized x-ray structures that fit most closely to the
data. The distributions look remarkably similar to those in Fig.
5A, with the peak in the distribution for the cAMP-ligated
CRP in the closed form occurring at a lower value than that for the
nonligated CRP in the open form.

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Fig. 5.
Distance distribution functions obtained from
the desmeared data for unligated CRP ( ) and cAMP-ligated CRP ( )
in D2O solvent (A) and from
energy-minimized x-ray structures (B) of unligated CRP in
the open form ( ) and cAMP-ligated CRP in the closed form ( )
(A).
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|
 |
DISCUSSION |
The SANS results show that ligation of CRP by cAMP apparently
induces a conformational change in CRP from the exclusively open form
in solution to the more contracted closed form, whereas CRP* remains
essentially unchanged in the presence of cAMP in solution. It should be
emphasized that the energy-minimized open form of unligated CRP is
based on energy minimization of the x-ray crystal structure of
cAMP-ligated CRP, exclusively in the open form, with cAMP removed,
because the x-ray crystal structure of unligated CRP is unavailable.
The good agreement between this conformation of unligated CRP and the
experimental SANS curve does imply that this energy-minimized open form
may indeed be the stable conformation of unligated CRP in solution.
These results agree with NMR measurements, which show a tightening up
of CRP into a more rigid protein upon cAMP binding (2). The magnitude of the change of approximately 1 Å in the radius of gyration also agrees with the 0.7 ± 0.1 Å shrinkage observed in the Stokes
radius of CRP upon ligation with cAMP (22). Although the change in CRP
is in agreement with earlier small angle x-ray scattering measurements,
which also indicate a contraction in the shape of CRP upon binding of
cAMP, the x-ray measurements exhibited a larger change in
Rg from 29 Å for unligated CRP to 25 Å for the
cAMP-ligated CRP complex (5). Calculations on the energy-minimized
unligated and ligated CRP structures show that
Rg would contract only from 20.5 Å for the open
form of CRP to 19.2 Å for the closed form of the complex. The x-ray
scattering results show not only a large change in
Rg, but also an absolute
Rg value 6-9 Å larger than could be calculated
from the x-ray crystallographic structures. The presence of aggregates
in the small angle x-ray scattering samples could easily account for
the higher Rg values and larger relative change
in Rg in the presence of cAMP.
The change to the closed form for the cAMP-ligated CRP complexes in
solution also agrees with molecular dynamics simulations on the
cAMP-ligated CRP complex, which predict that the closed form is the
more energetically stable form of this complex in solution (9). In
addition, the x-ray crystallographic structure of the cAMP-ligated CRP
complexed with DNA shows that the cAMP-ligated CRP exists exclusively
in the closed form for this ternary complex (8). Lack of growth for CRP
crystals in the absence of cAMP may arise from the CRP in solution
being exclusively in the open form.
The energy-minimized structures for unligated CRP in the open form and
cAMP-ligated CRP in the closed form are shown in Fig. 6, which illustrates the small, but
significant, structural differences between the two forms. A model
proposed by Garges and Adhya (23) for the conformational change of CRP
upon cAMP binding involves four structural changes in CRP: 1) a change
in the orientation of the D
-helix (Fig. 6), 2) an interaction
between the cAMP and DNA binding domains, 3) re-alignment of the CRP
monomers, and 4) more importantly, the "pushing away" of the
DNA-binding F
-helices (Fig. 6) from the protein. These changes also
allow the two F
-helices to bind into two adjacent grooves of DNA. The changes described by the model with respect to the interaction between the cAMP and DNA binding domains and a change in the distance between the DNA-binding F
-helices are also shown in Fig. 6 in the
transition from the unligated open form of CRP (Fig. 6A) to the ligated closed form of CRP (Fig. 6B). The distance
between the F
-helices in the closed form is more conducive to DNA
binding as evident by the cAMP-CRP-DNA crystal structure, which is
exclusively in the closed form. Thus, the changes described in this
model support the SANS results, which show a conformational change from unligated CRP in exclusively the open form to the closed form upon cAMP
ligation in solution.

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Fig. 6.
Energy-minimized structure for nonligated CRP
in the open form (A) and for cAMP-ligated CRP in the closed
form (B).
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The lack of significant changes in the CRP* scattering curves upon
complexation with cAMP implies that CRP* undergoes little, or possibly
no, structural change in the presence of cAMP. This is in agreement
with the observation that transcription is activated in the absence of
cAMP in cells which contain CRP* (11), implying that unligated CRP* is
already in the correct conformation for binding of the DNA. To
substantiate that this conformation is the closed form, fits of the
energy-minimized structure of CRP* to the SANS data must await the
determination of a good starting structure for CRP* from x-ray
crystallography.