Proton nuclear magnetic resonance studies on cyclic nucleotide binding to the Escherichia coli adenosine cyclic 3',5'-phosphate receptor protein.
Proton nuclear magnetic resonance studies on cyclic nucleotide binding to the Escherichia coli adenosine cyclic 3',5'-phosphate receptor protein.
复制标题
环核苷酸与大肠杆菌腺苷环 3,5-磷酸受体蛋白结合的质子核磁共振研究。
作者：
A. Gronenborn;G. Clore
本文报道了大肠杆菌环腺苷3 ′，5 ′-磷酸受体蛋白（CRP）及其与环腺苷3 ′，5 ′-磷酸（CAMP）、环鸟苷3 ′，5 ′-磷酸（cGMP）和一些类似物的复合物的~ 1H NMR研究。观察到五组咪唑质子共振，对应于通过氨基酸分析发现的每个亚基五个组氨酸（安德森，W. B.，施耐德A. B.，Emmer，M.，帕尔曼河L.，& Pastan，I.（1971）J.Biol.Chem.246，592949371，四个组氨酸残基A、B、C和D的特征在于咪唑质子共振，对于分子量为45 000的蛋白质具有异常窄的谱线宽度（3-5 Hz）和6-7的pK，这表明它们位于蛋白质的移动的区域。相比之下，第五个组氨酸残基E具有宽C（2）质子共振（-15 Hz）和非常低的pK（55），表明它以去质子化状态埋藏在蛋白质的刚性部分中。向CRP中添加环核苷酸不会导致蛋白质谱的任何剧烈变化。最显著的变化与cAMP的加入有关，其特征在于蛋白质的所有质子共振谱的整体加宽和芳香区高场端的显著变化。加入cAMP和cGMP后，可以检测到CRP结构的缓慢构象变化。这种构象转变发生在环核苷酸分子与结合位点之一结合之后，并且仅在两个环核苷酸结合位点几乎完全饱和时才完成。对于cGMPCRP复合物，两种构象之间的相互转化率的上限为18 s-1，而对于cAMP·CRP复合物，相互转化率的范围为60-120 s-1。用转移核Overhauser增强法研究了与CRP结合的环核苷酸的糖苷键构象，结果表明cAMP及其类似物以顺式构象结合，糖苷键扭转角x（0（4 ′）-C（1 ′）-N（9）-C（4））在45-55 ′之间，cGMP及其类似物环肌苷3 ′，5 '-磷酸（cIMP）以反构象结合，x值分别为225'和240°。这与存在于顺式/反式平衡混合物中的游离环核苷酸的情况形成对比，cAMP主要处于反式构象，cGMP主要处于顺式构象。CRP的构象选择的影响和可能的机制进行了讨论。许多结合的环核苷酸（包括cAMP和cGMP）的糖苷键。我们发现，而cAMP和它的类似物是结合在顺式构象，cGMP和它的类似物是结合在反构象。这与它们在自由溶液中的构象形成对比，
A 'H NMR study on the Escherichia coli aden- osine cyclic 3',5'-phosphate receptor protein (CRP) alone and in its complexes with adenosine cyclic 3',5'-phosphate (CAMP), guanosine cyclic 3',5'-phosphate (cGMP), and a number of analogues is presented. Five sets of imidazole proton reso- nances are seen, corresponding to the five histidines per subunit found by amino acid analysis (Anderson, W. B., Schneider, A. B., Emmer, M., Perlman, R. L., & Pastan, I. (1971) J. Biol. Chem. 246, 592949371, Four of the histidine residues, A, B, C, and D, are characterized by imidazole proton resonances with unusually narrow line widths (3-5 Hz) for a protein of molecular weight 45 000 and pKs in the range 6-7, suggesting that they lie in mobile regions of the protein. In contrast, the fifth histidine residue, E, has a broad C(2) proton resonance (- 15 Hz) and a very low pK (55), indicating that it is buried in the deprotonated state in a rigid portion of the protein. The addition of cyclic nucleotides to CRP does not lead to any drastic changes in the protein spectrum. The most prominent changes are associated with the addition of cAMP and are characterized by an overall broadening of all the proton res- onances of the protein and marked changes at the high-field end of the aromatic region. Following the addition of cAMP and cGMP, a slow conformational change in the structure of CRP can be detected. This conformational transition occurs subsequent to the binding of a cyclic nucleotide molecule to one of the binding sites and is only complete when both cyclic nucleotide binding sites are almost completely saturated. For the cGMPCRP complex, the upper limit for the intercon- version rate between the two conformations is 18 s-I, and for the cAMP.CRP complex, the interconversion rate lies in the range 60-120 s-'. The conformations about the glycosidic bond of cyclic nucleotides bound to CRP were investigated by the measurement of transferred nuclear Overhauser en- hancements, which showed that cAMP and its analogues are bound in the syn conformation with values for the glycosidic bond torsion angle x (0(4')-C( l')-N(9)-C(4)) in the range 45-55' and that cGMP and its analogue inosine cyclic 3',5'-phosphate (cIMP) are bound in the anti conformation with values of x of 225' and 240°, respectively. This contrasts to the situation in free cyclic nucleotides which exist in a syn/anti equilibrium mixture, cAMP being predominantly in the anti conformation and cGMP predominantly in the syn conformation. The implications and possible mechanism of this conformational selection by CRP are discussed. the glycosidic bond of a number of bound cyclic nucleotides including cAMP and cGMP. We show that whereas cAMP and its analogous are bound in the syn conformation, cGMP and its analogues are bound in the anti conformation. This contrasts with their conformations in free solution where cyclic
大肠杆菌环 3,5-磷酸腺苷受体蛋白的氨基和羧基近端片段的分离和表征。
影响因子：
2.9
作者：
Aiba,H;Krakow,JS
通讯作者：
Krakow,JS