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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。本研究旨在利用小角X射线散射技术研究钙调素及其相关激酶在与钙调素结合和捕获后的构象变化。多功能Ca 2 +/钙调蛋白依赖性激酶II（CaMKII）是一种寡聚体，广泛表达的丝氨酸/苏氨酸激酶，在许多细胞功能中发挥作用，包括细胞周期调节，蛋白质分泌，凋亡和基因表达。然而，CaMKII已经被研究得最多的是其在突触可塑性中的作用，特别是长时程增强。在这里，大量的证据表明CaMKII是调节突触强度的关键分子之一。CaMKII在与Ca 2+结合的钙调蛋白（CaM）结合后被激活。如果刺激水平足够强，CaMKII在CaM结合后发生自磷酸化。这以未知的方式改变了酶的结构特性，使得CaMKII现在即使在去除Ca 2+后仍保持高水平的活性。此外，自磷酸化的第二个结果是CaM对CaMKII的亲和力由于CaMKII-CaM解离速率降低>1000倍而显著增加，这种现象称为CaM捕获。由于自磷酸化和/或CaM捕获，CaMKII现在保持活性足够长的时间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，将采用激酶的单体形式。数据最初将以Guinier图的形式进行分析，以提取所检查的特定颗粒的回转半径。还将检查对分布函数以提取关于每个颗粒的形状信息。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