Developing an assay for Polar Organizing Protein Z (PopZ) mutant screening
Hass, Allison Paige, Laura Vigil, Derek
Hass, Allison
Paige, Laura
Vigil, Derek
Abstract
Description
The long-standing model that protein structure is critical to function has been expanded to include intrinsically disordered proteins (IDPs). IDPs are present in all kingdoms of life, and they facilitate diverse biological functions. IDPs lack stable structures and exist as conformational ensembles, making the study of IDP structure-function relationships particularly challenging. In this project, our approach is to perform IDP structure-function analyses in a relatively efficient manner, by leveraging the inherent experimental advantages of a bacterial system. The focus of our studies is a bacterial IDP called PopZ, which is responsible for creating signaling networks at the cell poles in Alphaproteobacteria. In our experiments, we employ recombinant E. coli strains that co-express genetic variants of PopZ together with a known binding partner protein. Both proteins are tagged with a fluorescent protein for the purpose of subcellular localization by fluorescence microscopy. In our experiments, liquid cultures were inoculated, diluted, and recombinant protein expression was induced with IPTG and arabinose. As expected, wildtype PopZ exhibited polar localization, and the GFP-tagged binding partners, ChpT and ParB, co-localized with the polar PopZ foci. When a mutant form of PopZ was investigated in this experiment, it localized to cell poles but the binding partners failed to co-localize, indicating a defect in protein-protein interaction. These pilot experiments, together with related controls, verified the effectiveness and accuracy of the assay. They also provided information for creating detailed protocols for future PopZ mutant screening, which will be carried out in the summer of 2017 at UW-Casper.
Date
Journal Title
Journal ISSN
Volume Title
Publisher
University of Wyoming Libraries
Research Projects
Organizational Units
Journal Issue
Keywords
Education