This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Neurotoxicity in Alzheimer disease is attributed to proteolytic fragments of amyloid precursor protein (APP). The carboxy terminal fragments of APP (CTFs) have been found in AD patients` brain and reported to exhibit higher neurotoxicity than beta-amyloid. The CTFs are shown to be in the center of a complex protein-protein network involving interactions with phosphotyrosine binding (PTB) domain and Src homology 2 (SH2) domain containing proteins. Fe65, one of the cytoplasmic neural protein having PTB and WW domains, interacts with APP-CTFs and translocate into the nucleus. It is important to determine the atomic structures of different domains of the adopter-like protein, Fe65, to understand the role of these domains in protein-protein interaction and also in nuclear translocation of APP-CTFs and subsequent transcription activation. One of the interaction domains, a small 38 amino acid peptide called WW domain was over-expressed in E.coli. The purified protein was crystallized in native form as well as co-crystallized with short peptides from their binding partners. Because of the long c axis, we could collect only 3 & data set at in-house diffractometer, even though the crystals diffract to better than 2.5 & resolution. The Se-Met derivative crystallizes under identical condition as native crystals and 5 mM DTT and reducing agent. Se-Met crystals grow to approximately 250x100x100 microns in 3-7 days. Synchrotron beam-time at a MAD line is essential for us to solve this important protein domain by collecting MAD data set for the Se-Met crystal and also high resolution native data sets for the co-crystals with different peptides. The structure will be solved by Se-MAD.
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