Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface

Praopim Limsakul, Qin Peng, Yiqian Wu, Molly E. Allen, Jing Liang, Albert G. Remacle, Tyler Lopez, Xin Ge, Brian K. Kay, Huimin Zhao, Alex Y. Strongin, Xiang Lei Yang, Shaoying Lu, Yingxiao Wang

Research output: Contribution to journalArticle

Abstract

Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells. Limsakul et al. demonstrate that directed evolution and sequence-function analysis are promising tools for engineering molecular binders and hybrid FRET biosensors, which reveal new distinct subcellular features of MT1-MMP molecular regulations at the extracellular surface of live cells.

Original languageEnglish (US)
Pages (from-to)370-379.e4
JournalCell chemical biology
Volume25
Issue number4
DOIs
StatePublished - Apr 19 2018

Keywords

  • FRET biosensor
  • MT1-MMP
  • R-phycoerythrin
  • cell-cell contacts
  • directed evolution
  • monobody

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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  • Cite this

    Limsakul, P., Peng, Q., Wu, Y., Allen, M. E., Liang, J., Remacle, A. G., Lopez, T., Ge, X., Kay, B. K., Zhao, H., Strongin, A. Y., Yang, X. L., Lu, S., & Wang, Y. (2018). Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface. Cell chemical biology, 25(4), 370-379.e4. https://doi.org/10.1016/j.chembiol.2018.01.002