General Anesthetic-Protein Interactions:Protein Kinase C

描述（由申请人提供）： 在美国，每年约有2500万患者使用具有非常低的治疗指数的全身麻醉剂进行手术，其分子机制仍然未知，阻碍了改进药物的设计。拟议研究的广泛长期目标是确定影响蛋白质功能的全身麻醉剂结合位点的分子决定因素。待研究的特定蛋白质是蛋白激酶C（PKC）。已知挥发性麻醉剂和长链醇影响活化的PKC的活性。我们的目的是测试的假设，麻醉剂作用于调节域（C1）的两个二酰基甘油（DAG）的结合位点（C1 A和B）定位全身麻醉剂结合位点的PKC光标记他们与最近开发的光活化麻醉剂醇。将通过质谱法鉴别光标记残留物。令人鼓舞的初步结果表明，这些药物与分离的第二个富含半胱氨酸（C1 B）的PKC 6的调节亚结构域在C1 B佛波醇结合位点附近的酪氨酸相互作用，并且C1 A片段表现出不同的结合特性。C1 B片段的晶体结构已经公布，我们将确定其与各种全身麻醉剂结合的结构。将在该结合口袋内进行设计突变，以确定在分子水平上管理麻醉剂-蛋白质相互作用的原则。将对C1 A进行类似的研究。在完整的C1结构域中，我们将测试C1 A和C1 B亚结构域之间的相互作用有助于麻醉剂结合位点的假设。麻醉剂和DAG位点在C1结构域中C1 A和C1 B处的变构相互作用的重要性将通过使其与和不与佛波酯结合的复合物以及在存在和不存在麻醉剂的情况下结晶来评估。将通过平行测量麻醉剂和phorboI-C1相互作用来解释结构变化。

DESCRIPTION (provided by applicant): In the USA approximately 25 million patients/year undergo surgery using general anesthetics having very low therapeutic indices and whose molecular mechanisms remain unknown, hampering the design of improved agents. The broad long-term objective of the proposed research is to define the molecular determinants of those general anesthetic binding sites that effect the function of proteins. The specific protein to be studied is protein kinase C (PKC). Volatile Anesthetics and long chain alcohols are known to affect the activity of activated PKC. We aim to test the hypothesis that anesthetics act on the regulatory domain (C1) at the two diacylglycerol (DAG) binding sites (C1A & B) by locating general anesthetic binding sites on PKC by photolabeling them with recently developed photoactivatable anesthetic alcohols. Photolabeled residues will be identified by mass spectrometry. Encouraging preliminary results show these agents to interact with the isolated second cysteine-rich (C1B) regulatory subdomain of PKC6 at a tyrosine adjacent to the C1B phorbol binding site and that the C1A fragment exhibits different binding characteristics. The crystal structure of the C1B fragment has been published, and we will determine its structure with various general anesthetics bound. Designed mutations within this binding pocket will be made in order to determine the principles governing anesthetic-protein interactions at the molecular level. Similar studies will be undertaken with C1A. In the intact C1 domain, we will test the hypothesis that interactions between the C1A and C1B subdomains contribute to the anesthetic binding sites. The importance of allosteric interactions between the anesthetic and DAG sites at C1A & C1B in the C1 domain will be assessed by crystallizing it complexed with and without a phorbol ester bound and in the presence and absence of anesthetics. Structural changes will be interpreted by measuring anesthetic and phorboI-C1 interactions in parallel.