472412 Design and Evaluation of Engineered Protein Biosensors for Live Cell Imaging of Epidermal Growth Factor Receptor Phosphorylation

Monday, November 14, 2016: 9:30 AM
Continental 8 (Hilton San Francisco Union Square)
Shoeb Ahmed, Bangladesh University of Engineering and Technology, Dhaka, Bangladesh, Karthik Tiruthani, North Carolina State University, Raleigh, NC, Jason Haugh, Chemical & Biomolecular Engineering, North Carolina State University, Raleigh, NC and Balaji Rao, Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC

Live-cell imaging studies can provide mechanistic insight into dynamics of receptor-mediated cell signaling; however the availability of intracellular biosensors is presently limited. The tandem SH2 domain from PLCγ1 (tSH2-WT) has been used as a marker of phosphorylated epidermal growth factor receptor (EGFR). Consistent with the promiscuity of SH2 domains, we show that tSH2-WT lacks specificity for phosphorylated EGFR. Further, EGF-stimulated recruitment of tSH2-WT differs qualitatively from the expected kinetics of EGFR phosphorylation. Analysis of a mathematical model shows that the high avidity of tSH2‑WT, because of sequential binding of both SH2 domains, explains the discrepancy between the actual and expected biosensor readout. To address the limitations of the tSH2-WT biosensor, we constructed a combinatorial library through random mutagenesis of the C-terminal SH2 domain (cSH2) of PLCγ1. The library was screened using yeast surface display to isolate a mutant protein (mSH2) with enhanced specificity for the Y992 phosphorylation site in EGFR (pY992). Accordingly, a biosensor based on mSH2 faithfully reports kinetics of EGFR phosphorylation in live-cell imaging experiments. This approach, integrating theoretical considerations with a protein engineering strategy, can be generalized to design and evaluate suitable biosensors for specific intracellular targets.

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See more of this Session: Biomolecular Engineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division