Nitric oxide synthase mechanism and calmodulin dependent activation

一氧化氮合酶机制和钙调蛋白依赖性激活

• 批准号: 183521-2012
• 负责人: Guillemette, Guy
• 金额: $ 2.48万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=569636
• 关键词: Nitric; oxide; synthase; mechanism; calmodulin

Nitric oxide (NO) is an important endogenous messenger in a variety of physiological and pathophysiological processes. NO is synthesized by the three isoforms of an enzyme, nitric oxide synthase (NOS) that are in part controlled by the binding of calmodulin (CaM). CaM is the primary protein mediator of the calcium cation which is also an important endogenous secondary messenger. CaM undergoes calcium concentration dependent conformational changes that allow it to differentially bind and activate scores of target proteins including the three NOS isozymes and voltage-gated calcium channels (VGCCs). Exactly how CaM manages to bind and sometimes control the activity of such a large variety of proteins and enzymes is not fully understood. The long-term objectives of this research program are to further our understanding of: 1) the mechanism and control of NO synthesis by NOS; 2) the CaM-dependent activation of its numerous target proteins including NOS isozymes and VGCCs. How CaM controls electron transfer and in turn NOS activity is not fully understood. One of our objectives during the course of this granting period is to investigate the dynamics and processes by which CaM interacts and controls electron transfer in all three NOS isoforms. VGCCs are also modulated by calcium-dependent feedback mechanisms that are modulated by CaM. How exactly CaM is implicated in these processes is not well known and will also be the subject of our investigation. The students that join our interdisciplinary research team will be trained in a wide variety of methods including recombinant DNA and protein techniques to produce and modify different forms of the enzymes needed for our investigations. Several biophysical techniques will be used in our investigations including visible, fluorescence and nuclear magnetic resonance spectroscopy, as well as calorimetry and mass spectrometry.

一氧化氮（NO）是一种重要的内源性信使，参与多种生理和病理生理过程。NO由一氧化氮合酶（NOS）的三种同工型合成，其部分由钙调蛋白（CaM）的结合控制。 CaM是钙离子的主要蛋白质介导物，也是重要的内源性第二信使。 CaM经历钙浓度依赖性构象变化，使其能够差异结合和激活靶蛋白，包括三种NOS同工酶和电压门控钙通道（VGCC）的分数。 钙调素究竟是如何设法结合，有时控制这样一个大的各种蛋白质和酶的活性是不完全理解。 本研究计划的长期目标是进一步了解：1）NOS合成NO的机制和控制; 2）CaM依赖性激活其众多靶蛋白，包括NOS同工酶和VGCC。 钙调素如何控制电子传递，进而控制一氧化氮合酶的活性还不完全清楚。我们的目标之一，在这个授予期间的过程中，是调查的动力学和过程中，钙调素相互作用和控制电子转移在所有三个NOS亚型。 VGCC也受钙调素调节的钙依赖性反馈机制调节。 究竟钙调素是如何牵连在这些过程中并不为人所知，也将是我们的调查的主题。 加入我们跨学科研究团队的学生将接受各种方法的培训，包括重组DNA和蛋白质技术，以生产和修改我们研究所需的不同形式的酶。 几种生物物理技术将用于我们的调查，包括可见光，荧光和核磁共振光谱，以及量热法和质谱法。