Energetics of intersubunit and intrasubunit interactions of Escherichia coli adenosine cyclic 3',5'-phosphate receptor protein.


Abstract

Escherichia coli cAMP receptor protein (CRP) regulates the expression of a large number of catabolite-sensitive genes. The mechanism of CRP regulation most likely involves communication between subunits and domains. A specific message, such as the activation of CRP, may be manifested as a change in the interactions between these structural entities. Hence, the elucidation of the regulatory mechanism would require a quantitative evaluation of the energetics involved in these interactions. Thus, a study was initiated to define the conditions for reversible denaturation of CRP and to quantitatively assess the energetics involved in the intra- and intersubunit interactions in CRP. The denaturation of CRP was induced by guanidine hydrochloride. The equilibrium unfolding reaction of CRP was monitored by three spectroscopic techniques, namely, fluorescence intensity, fluorescence anisotropy, and circular dichroism. The spectroscopic data implied that CRP unfolds in a single cooperative transition. Sedimentation equilibrium data showed that CRP is dissociated into its monomeric state in high concentrations of denaturant. Unfolding of CRP is completely reversible, as indicated by fluorescence and circular dichroism measurements, and sedimentation data indicated that a dimeric structure of CRP was recovered. The functional and other structural properties of renatured and native CRP have also been examined. Quantitatively identical results were obtained. Results from additional studies as a function of protein concentration and from computer simulation demonstrated that the denaturation of CRP induced by guanidine hydrochloride proceeds according to the following pathway: (CRP2)Native<-->2(CRP)Native<-->2(CRP)Denatured. The delta G values for dissociation (delta Gd) and unfolding (delta G(u)) in the absence of guanidine hydrochloride were determined by linear extrapolation, yielding values of 12.0 +/- 0.6 and 7.2 +/- 0.1 kcal/mol, respectively. To examine the effect of the DNA binding domain on the stability of the cAMP binding domain, two proteolytically resistant cAMP binding cores were prepared from CRP in the presence of cAMP by subtilisin and chymotrypsin digestion, yielding S-CRP and CH-CRP, respectively. Results from an equilibrium denaturation study indicated that the denaturation of both CH-CRP and S-CRP is also completely reversible. Both S-CRP and CH-CRP exist as stable dimers with similar delta Gd values of 10.1 +/- 0.4 and 9.5 +/- 0.4 kcal/mol, respectively. Results from this study in conjunction with crystallographic data [McKay, D. B., Weber, I. T., & Stietz, T. A. (1982) J. Biol. Chem. 257, 9518-9524] indicate that the DNA binding domain and the C-helix are not the only structural elements that are responsible for subunit dimerization.(ABSTRACT TRUNCATED AT 400 WORDS) Study holds ProTherm entries: 4555 Extra Details: additive : EDTA(1 mM), regulatory mechanism; energetics; cooperative transition;,subunit dimerization

Submission Details

ID: QEatGfPi

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Cheng X;Gonzalez ML;Lee JC,Biochemistry (1993) Energetics of intersubunit and intrasubunit interactions of Escherichia coli adenosine cyclic 3',5'-phosphate receptor protein. PMID:8394127
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
6B6H 2017-10-02T00:00:00+0000 3.9 The cryo-EM structure of a bacterial class I transcription activation complex
6PB4 2019-06-13T00:00:00+0000 4.35 The E. coli class-II CAP-dependent transcription activation complex with de novo RNA transcript at the state 2
6PB5 2019-06-13T00:00:00+0000 4.52 The E. coli class-II CAP-dependent transcription activation complex at the state 1 architecture
6PB6 2019-06-13T00:00:00+0000 4.29 The E. coli class-II CAP-dependent transcription activation complex at the state 2
1CGP 1991-08-12T00:00:00+0000 3.0 CATABOLITE GENE ACTIVATOR PROTEIN (CAP)/DNA COMPLEX + ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
1G6N 2000-11-07T00:00:00+0000 2.1 2.1 ANGSTROM STRUCTURE OF CAP-CAMP
1HW5 2001-01-09T00:00:00+0000 1.82 THE CAP/CRP VARIANT T127L/S128A
1I5Z 2001-03-01T00:00:00+0000 1.9 STRUCTURE OF CRP-CAMP AT 1.9 A
1I6X 2001-03-06T00:00:00+0000 2.2 STRUCTURE OF A STAR MUTANT CRP-CAMP AT 2.2 A
1J59 2002-03-01T00:00:00+0000 2.5 CATABOLITE GENE ACTIVATOR PROTEIN (CAP)/DNA COMPLEX + ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.5 cAMP-activated global transcriptional regulator CRP P0A2T6 CRP_SALTY
99.5 cAMP-activated global transcriptional regulator CRP P0A2T7 CRP_KLEAE
100.0 cAMP-activated global transcriptional regulator CRP P0ACK1 CRP_SHIFL
100.0 cAMP-activated global transcriptional regulator CRP P0ACJ8 CRP_ECOLI
100.0 cAMP-activated global transcriptional regulator CRP P0ACJ9 CRP_ECOL6
100.0 cAMP-activated global transcriptional regulator CRP P0ACK0 CRP_ECO57