SOLUTION STRUCT OF PHOSPHORYLATED FORM OF TWO COMPONENT RESPONSE REGULATOR NTRC

双组分调节系统是最常见的一种 在细菌中的信号转导机制，最近已被 发现于真核生物中。 在这些系统中，组氨酸激酶 自身磷酸化是对环境刺激的反应， 为响应调节剂提供磷酸盐，随后， 将信号传导至下游靶标。 没有人知道 反应调节因子激活的结构基础， 磷酸化，因为磷酸化蛋白的寿命是 对结构分析来说是个很短的词 我们使用 反应调节剂，NTRC，它控制氮代谢，作为一种 模型系统 未磷酸化形式的结构已经被 在我们的实验室中测定（Bullman，等人，Biochemistry 34，1413-1424）。 我们最近获得了维持磷酸化的条件， 状态足够长以通过NMR确定结构。 这是通过 使用小分子作为 磷酸供体。 然而，由于聚集和快速周转， 蛋白质浓度限于0.5mM。 更高的灵敏度将大大有助于测定 750 MHz磁铁和8 mm探头的组合。 HSQC是在我们实验室拍摄的 表明磷酸化后的构象变化涉及 仅分子的一部分，使得已知的NMR数据 未磷酸化的形式将有助于分析活化的， 磷酸化形式。 这项工作应该提供第一个例子， 通过磷酸化激活的结构理解， 反应调节子超家族。 鉴于高序列和 两组分系统之间的结构同源性 NTRC中结构/功能关系对于其他 响应调节器。 此外，蛋白质的结构变化 由磷酸化引发的是令人感兴趣的，因为这是 用于调节蛋白质的最常见的共价修饰 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.