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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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/钙调蛋白依赖性激酶II（CaMKII）是已知在学习和记忆中发挥重要作用的12个单体的非常大（640 kDa）的激酶组装体。 它通常处于完全失活状态，其所有12种激酶仅显示最低限度的活性。 暴露于钙结合的钙调蛋白释放激酶活性，然而，该过程的分子机制的细节已经不可能从生物化学研究中诱导。了解激酶的调节机制将需要激酶的活性和非活性形式的详细分子模型。最近，我们从C. elegans CamKII直向同源物。在ALS进行的初步衍射实验表明，这些晶体在某些方向上各向同性地将X射线吸收到~7.5，并微弱地吸收到3.9。对晶胞和非晶体学对称元素的初步分析表明，晶体在晶胞中含有12个CaMKII分子（约640 kDa）的完整全酶，但数据非常弱，其质量不足以进行进一步分析。 （2）DNA复制。(i)DNA聚合酶是负责复制染色体的酶。来自不同生物体的几种聚合酶的结构已经确定，然而，主要的E.大肠杆菌复制型DNA聚合酶至今仍然难以捉摸。最近，我们获得了E.大肠杆菌聚合酶催化亚单位。晶体很小（约50微米），对X射线的吸收很弱，约为4微米。

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¿ .