STRUCTURAL STUDIES OF CALCIUM/CALMODULIN DEPENDENT KINASE II AND E COLI REPLICA

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。本研究涉及两类蛋白质复合物，其中已获得关键组件的晶体。(1)钙/钙调素依赖性激酶II （CaMKII）是一个非常大的（640 kDa）的激酶组件，由12个单体组成，已知在学习和记忆中起重要作用。它通常处于完全失活状态，其所有12种激酶仅表现出最低限度的活性。暴露于钙结合的钙调素释放激酶活性，然而这一过程的分子机制的细节已经不可能从生化研究诱导。了解激酶的调节机制将需要激酶活性和失活形式的详细分子模型。最近，我们获得了秀丽隐杆线虫CamKII同源物的结晶。在ALS进行的初步衍射实验表明，这些晶体在某些方向上衍射x射线各向同性至~7.5°c，弱至3.9°c。对单位细胞和非晶体对称元素的初步分析表明，晶体在单位细胞中含有一个完整的12个CaMKII分子（约640kDa）的全酶，但数据非常弱，其质量不足以允许进一步分析。(2) DNA复制。DNA聚合酶是负责染色体复制的酶。来自不同生物体的几种聚合酶的结构已经确定，然而，大肠杆菌的主要复制DNA聚合酶的结构迄今为止仍然是难以捉摸的。最近，我们获得了大肠杆菌聚合酶催化亚基的晶体。晶体很小（~50¿¿m），对x射线的衍射弱至~4¿¿。

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 research is concerned with two classes of protein complexes in which crystals of key assemblies have been obtained. (1) The Calcium/Calmodulin dependent kinase II (CaMKII) is a very large (640 kDa) kinase assembly of 12 monomers known to play an important role in learning and memory. It is normally held in a fully inactive state, with all 12 of its kinases showing only minimal activity. Exposure to calcium bound calmodulin releases the kinase activity, however the details of the molecular mechanism of this process have been impossible to induce from biochemical studies. Understanding the regulatory mechanism of the kinase will require a detailed molecular model of both active and inactivate forms of the kinase. Recently, we have obtained crystals from the C. elegans CamKII orthologue. Initial diffraction experiments performed at the ALS indicate that these crystals diffract x-rays isotropically to ~7.5 ¿¿ and weakly to 3.9 ¿¿in certain directions. Preliminary analysis of the unit cell and non-crystallographic symmetry elements indicates that the crystals contain an intact holoenzyme of 12 CaMKII molecules (about 640kDa) in the unit cell, however the data is very weak and its quality is not sufficient to allow for further analysis. (2)DNA Replication. (i) DNA polymerase is the enzyme responsible for replicating chromosomes. The structures of several polymerases from various organisms have been determined, however, the structure of the major E. coli replicative DNA polymerase has thus far remained elusive. Recently, we obtained crystals of the E. coli polymerase catalytic subunit. The crystals are small (~50 ¿¿m), and diffract x-rays weakly to ~4¿¿.