General Anesthetic-Protein Interactions:Protein Kinase C

全身麻醉药-蛋白质相互作用：蛋白激酶 C

• 批准号: 6710963
• 负责人: KEITH W MILLER
• 金额: $ 30.87万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6710963
• 关键词: alcohols; allosteric site; aminoacid; binding sites; biological signal transduction; circular dichroism; crystallization; diacylglycerols; electrospray ionization mass spectrometry; general anesthesia; high performance liquid chromatography; intermolecular interaction; mass spectrometry; protein kinase C; protein purification; protein structure function; proteolysis; site directed mutagenesis; stoichiometry

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.

描述（由申请人提供）： 在美国，每年约有 2500 万患者接受全身麻醉剂的手术，这些麻醉剂的治疗指数非常低，而且其分子机制仍不清楚，这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。要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会影响激活的 PKC 的活性。我们的目的是通过用最近开发的光活化麻醉醇进行光标记来定位 PKC 上的全身麻醉剂结合位点，从而测试麻醉剂作用于两个二酰基甘油 (DAG) 结合位点（C1A 和 B）的调节域 (C1) 的假设。光标记的残留物将通过质谱法进行鉴定。令人鼓舞的初步结果表明，这些药物可与 PKC6 中分离的第二个富含半胱氨酸 (C1B) 的调节亚结构域相互作用，该亚结构域位于 C1B 佛波醇结合位点附近的酪氨酸处，并且 C1A 片段表现出不同的结合特征。 C1B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，以确定分子水平上控制麻醉剂-蛋白质相互作用的原理。类似的研究也将针对 C1A 进行。在完整的 C1 结构域中，我们将测试 C1A 和 C1B 子结构域之间的相互作用有助于麻醉剂结合位点的假设。 C1 结构域中 C1A 和 C1B 处的麻醉剂和 DAG 位点之间变构相互作用的重要性将通过在有或没有佛波酯结合和有或没有麻醉剂的情况下使其复合结晶来评估。将通过并行测量麻醉剂和佛波I-C1 相互作用来解释结构变化。