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Development of split-protein-based platforms for applications in chemical biology
Protein misfolding is implicated in numerous human diseases, including Alzheimer's disease, Parkinson's disease, and cystic fibrosis. Consequently, robust assays to rapidly assess protein solubility are essential for screening compounds that influence protein aggregation. Herein, we describe a set of self-assembling nanoluciferase (Nluc) fragments that produce A luminescent readout that is proportional for the amount of the N-terminal nanoluc fragment available for reassembly. Leveraging this observation we demonstrate the ability to assess protein solubility by fusing a protein of interest (POI) to the N-terminus of the N-terminal fragment of NLuc. The resulting luminescence of living bacteria is directly proportional to the solubility of the POI. This approach offers a novel, genetically encodable luminescent readout for the aggregation of disease-associated proteins in living cells. To demonstrate this approach we assessed the influence of mutations known to disrupt amyloid-beta 42 (A?42) aggregation, as well as inhibitors of protein aggregation. Additionally, we apply this sensitive platform to report on the influence of mutations as well as small molecules on amylin and polyQ aggregation. Additionally, we are interested in expanding split-protein reassembly to allow for the conditional control of cellular signaling pathways. The 150 human small GTPases are established regulators of important signaling pathways, including cell morphogenesis, cell division, and cell migration. Consequently, the ability to directly modulate small GTPase activity and assess cellular phenotypes would greatly enhance our molecular understanding of disease. Based on the principle that split-protein fragments are capable of refolding to their original structure in a concentration-induced manner, we have dissected a model small GTPase (Cdc42) within every loop of the protein tertiary structure. The resulting library of Cdc42 fragments were attached to the well-characterized rapamycin-dependent FKBP and FRB dimerization domains. Upon the addition of the small molecule rapamycin, Cdc42 activity was restored in a rapamycin-dependent manner. The fragments identified from this study provide tunable control over Cdc42 function and could be employed with user-defined protein-protein interaction domains. In the long term, the structural similarity between members of the small GTPase family may make it possible to apply the dissection sites identified in this work to other small GTPases in order to construct orthogonal networks of fragmented small GTPases.
Zhao, Jia, "Development of split-protein-based platforms for applications in chemical biology" (2016). ETD collection for University of Nebraska - Lincoln. AAI10143333.