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. Release time is given for the spring semester for Dr. Chambers to work on the following project: ERM family members may play a role in tumorigenesis. A more distantly related protein, merlin, is a tumor suppressor and has been shown to cause the formation of Neurofibromatosis II, a benign brain tumor, upon mutation. Furthermore, it has been demonstrated that expression of ERMs is often upregulated in various tumors. Only a single ERM homologue is present in many lower organisms, C. elegans, as well as a very divergent merlin. The C. elegans ERM homologue's DNA sequence is ~61% identical to the human ERMs while the merlin homologue is only 46% identical to human ERMs and merlin. To understand if this large variation changes the properties of these proteins I propose to: 1. Express and purify the full-length proteins and separately expressed domains with N-terminal cleavable tags. 2. Perform blot overlay and bead binding analysis to determine the nature of the FERM/C-terminus interaction. 3. Perform gel filtration on the purified proteins to compare conformations of the species. 4. Confirm the existence of an F-actin binding site in the ERM but not in merlin by F-actin cosedimentation. 5. Identify possible binding partners for the FERM domain of each species with affinity chromatography utilizing immobilized FERM domain and whole organism extracts. 6. Attempt to crystallize multiple protein products for collaborative studies with other laboratories. 7. Produce, characterize, and affinity purify antibodies raised to each protein.
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