SOLUTION STRUCT OF PHOSPHORYLATED FORM OF TWO COMPONENT RESPONSE REGULATOR NTRC

双重监管体系是最常见的监管体系之一 细菌中的信号转导机制，最近已经 发现于真核生物中。在这些系统中，一种组氨酸激酶 对环境刺激的反应是自动磷酸化， 为响应调节器提供磷酸盐，随后， 将信号传送到下游目标。对此一无所知 反应调节器被激活的结构基础 因为磷酸化蛋白质的寿命是 结构分析短得令人望而却步。我们使用的是 反应调节剂，NTRC，控制氮代谢，作为一种 模型系统。非磷酸化形式的结构一直是 由我们实验室测定(Volkman等人，《生物化学》34,1413-1424)。 我们最近获得了维持磷酸化的条件 状态足够长，可以通过核磁共振确定结构。此操作由以下人员完成 以小分子为基础建立稳态平衡 磷光。然而，由于聚合和快速的周转， 蛋白质浓度被限制在0.5 mM。因此，结构 更高的灵敏度将极大地促进测定 一个750兆赫的磁铁和一个8毫米的探头。HSQC是在我们实验室拍摄的 表明磷酸化时的构象变化包括 只有分子的一部分，所以已知的核磁共振数据 非磷酸化形式将有助于分析被激活的， 磷酸化形式。这项工作应该提供第一个例子 对细胞内磷酸化激活的结构理解 响应调节器超家族。鉴于较高的顺序和 二组分体系的结构同源性， NTRC中的结构/功能关系应适用于其他 应对监管机构。此外，蛋白质的结构变化 由磷酸化触发是很有趣的，因为这是 最常见的用于蛋白质调节的共价修饰 功能。

Two-component regulatory systems are one of the most common mechanisms for signal transduction in bacteria and have recently been found in eukaryotes. In these systems, a histidine kinase autophosphorylates in response to an envoironmental stimulus, providing the phoshate for the response regulator which, subsequently, transduces the signal to a downstream target. Nothing is known about the structural basis of the activation of response regulators upon phosphorylation because the lifetime of the phosphorylated protein is prohibitively short for structural analysis. We are using the response regulator, NTRC, which controls nitrogen metabolism, as a model system. The structure of the unphosphorylated form has been determined in our lab (Volkman, et al., Biochemistry 34, 1413-1424). We have recently obtained conditions which maintain the phosphorylated state long enough for structure determination by NMR. This is done by creating a steady state equilibrium using a small molecule as a phosphodonor. However, due to aggregation and fast turnover, the protein concentration is limited to 0.5 mM. Therefore, structure determination would be greatly facilitated by the higher sensitivity of a 750 MHz magnet and an 8 mm probe. HSQC's taken in our laboratory indicate that the conformational change upon phosphorylation involves only a portion of the molecule so that the known NMR data on the unphosphorylated form will be helpful in analyzing the activated, phosphorylated form. This work should provide the first example of structural understanding of activation via phosphorylation in the response regulator superfamily. Given the high sequence and structural homology among two-component systems, the structure/function relationships in NTRC should be general for other response regulators. Furthermore, the structural changes of a protein triggered by phosphorylation is of interest because this is one of the most common covalent modifications used for modulation of protein function.