Tài liệu Báo cáo khoa học: Steady-state and time-resolved fluorescence studies of conformational

Steady-state and time-resolved fluorescence studies of conformational

changes induced by cyclic AMP and DNA binding to cyclic AMP

receptor protein from Escherichia coli

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)2 complex leads to changes in the Trp13

microenvironment, whereas its binding in the CRP–

(cAMP)4 complex alters the surroundings of Trp85. Time￾resolved anisotropy measurements indicated that cAMP

binding in the CRP–(cAMP)2 complex 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)2 complex 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)4 complex

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)1 and 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 lM cAMP, 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: [email protected]

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)

Eur. J. Biochem. 270, 1413–1423 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03497.x