Yoshimura H, Hisabori T, Yanagisawa S, Ohmori M
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro, Tokyo 153-8902, Japan.
J Biol Chem. 2000 Mar 3;275(9):6241-5. doi: 10.1074/jbc.275.9.6241.
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.
对集胞藻PCC 6803编码与细菌cAMP受体蛋白的cAMP结合结构域同源结构域的三个开放阅读框进行了分析。这三个开放阅读框，即sll1371、sll1924和slr0593，分别命名为sycrp1、sycrp2和sypk，在大肠杆菌中作为His标签或谷胱甘肽S-转移酶融合蛋白表达并纯化，并对其生化特性进行了研究。使用这些重组蛋白进行平衡透析测量得到的结果表明，SYCRP1和SYPK对cAMP表现出结合亲和力，而SYCRP2则没有。His标签的SYCRP1对cAMP的解离常数约为3 microM。使用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺进行的交联实验表明，His标签的SYCRP1形成同源二聚体，cAMP的存在与否不影响同源二聚体的形成。氨基酸序列显示，SYCRP1在COOH末端区域具有与细菌cAMP受体蛋白的DNA结合结构域相似的结构域。与此一致的是，His标签的SYCRP1在cAMP存在的情况下与含有大肠杆菌cAMP受体蛋白共有序列的DNA形成复合物。这些结果强烈表明SYCRP1是一种新型的cAMP受体蛋白。
