STUDIES OF CONFORMATIONAL CHANGES IN CA2+/CALMODULIN DEPENDENT KINASE II USING S

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。这一建议是利用SAXS研究钙调素及其相关激酶在钙调素结合和俘获时发生的构象变化。多功能Ca2+/钙调蛋白依赖性激酶II （CaMKII）是一种寡聚物，广泛表达丝氨酸/苏氨酸激酶，在许多细胞功能中起作用，包括细胞周期调节，蛋白质分泌，细胞凋亡和基因表达。然而，CaMKII在突触可塑性，特别是长时程增强中的作用被研究得最多。大量证据表明，CaMKII是调节突触强度的关键分子之一。CaMKII在与Ca2+结合的钙调蛋白（CaM）结合时被激活。如果刺激水平足够强，CaMKII在与CaM结合时发生自磷酸化。这以一种未知的方式改变了酶的结构特性，使CaMKII现在即使在去除Ca2+时也保持高水平的活性。此外，自磷酸化的第二个结果是CaM对CaMKII的亲和力大大增加，这是由于CaMKII-CaM解离速率降低了1000倍，这种现象被称为CaM诱捕。由于自磷酸化和/或CaM捕获，CaMKII现在在足够的时间内保持活性，以启动改变突触强度的下游信号传导过程。为了便于使用SAXS检测CaMKII的结构变化，将采用该激酶的单体形式。数据将首先以吉尼尔图的形式进行分析，以提取被检查的特定粒子的旋转半径。还将检查对分布函数以提取每个粒子的形状信息。随后，将采用有助于构建低分辨率分子模型的算法，从散射数据生成三维密度图

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proposal is for using SAXS to study the conformational changes that occur in calmodulin and its associated kinase upon calmodulin binding and trapping. Multifunctional Ca2+/calmodulin dependent kinase II (CaMKII) is an oligomeric, widely expressed serine/threonine kinase that plays a role in many cellular functions including cell cycle regulation, protein secretion, apoptosis, and gene expression. However, CaMKII has been most studied in relation to its role in synaptic plasticity, and particularly long-term potentiation. Here, abundant evidence has implicated CaMKII as one of the key molecules that regulate the strength of synapses. CaMKII is activated upon binding of Ca2+-bound calmodulin (CaM). If the level of stimulation is sufficiently strong, CaMKII undergoes autophosphorylation upon CaM binding. This alters the structural properties of the enzyme in an unknown fashion such that CaMKII now retains a high level of activity even upon removal of Ca2+. Furthermore, a second result of autophosphorylation is that the affinity of CaM for CaMKII is substantially increased due to a >1000-fold decrease in the CaMKII-CaM dissociation rate, a phenomenon referred to as CaM trapping. Due to autophosphorylation and/or CaM trapping, CaMKII now remains active for a sufficient period of time to initiate the downstream signaling processes that alter synaptic strength. In order to facilitate detection of the structural changes in CaMKII using SAXS, a monomeric form of the kinase will be employed. The data will initially be analyzed in the form of a Guinier plot to extract the radius of gyration of the particular particle under examination. The pair distribution function will also be examined to extract shape information about each particle. Subsequently, algorithms that facilitate construction of low-resolution molecular models will be employed to generate three dimensional density maps from the scattering data