Identification and characterization of a novel cAMP receptor protein in the cyanobacterium Synechocystis sp. PCC 6803.
Three open reading frames of Synechocystis sp. PCC 6803 encoding a domain homologous with the cAMP binding domain of bacterial cAMP receptor protein were analyzed. These three open reading frames, sll1371, sll1924, and slr0593, which were named sycrp1, sycrp2, and sypk, respectively, were expressed in Escherichia coli as His-tagged or glutathione S-transferase fusion proteins and purified, and their biochemical properties were investigated. The results obtained for equilibrium dialysis measurements using these recombinant proteins suggest that SYCRP1 and SYPK show a binding affinity for cAMP while SYCRP2 does not. The dissociation constant of His-tagged SYCRP1 for cAMP is approximately 3 microM. A cross-linking experiment using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide revealed that His-tagged SYCRP1 forms a homodimer, and the presence or absence of cAMP does not affect the formation of the homodimer. The amino acid sequence reveals that SYCRP1 has a domain similar to the DNA binding domain of bacterial cAMP receptor protein in the COOH-terminal region. Consistent with this, His-tagged SYCRP1 forms a complex with DNA that contains the consensus sequence for E. coli cAMP receptor protein in the presence of cAMP. These results strongly suggest that SYCRP1 is a novel cAMP receptor protein.
Yoshimura
H
H
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro, Tokyo 153-8902, Japan.
Hisabori
T
T
Yanagisawa
S
S
Ohmori
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D000458:Cyanobacteria / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli; D008969:Molecular Sequence Data; D011485:Protein Binding; D011953:Receptors, Cyclic AMP / Q000737:chemistry* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016415:Sequence Alignment
D001426:Bacterial Proteins; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D011953:Receptors, Cyclic AMP; D011993:Recombinant Fusion Proteins; C405882:SYCRP1 protein, Synechocystis