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

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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¿¿.