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  3. Biosensors based on peptide exposure show single molecule conformations in live cells

Biosensors based on peptide exposure show single molecule conformations in live cells

  • Cell. 2021 Oct 28;184(22):5670-5685.e23. doi: 10.1016/j.cell.2021.09.026.
Bei Liu 1,  Orrin J Stone 1,  Michael Pablo 2,  J Cody Herron 3,  Ana T Nogueira 1,  Onur Dagliyan 1,  Jonathan B Grimm 4,  Luke D Lavis 4,  Timothy C Elston 5,  Klaus M Hahn 6
Affiliations

Affiliations

  • 1 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
  • 2 Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Program in Molecular and Cellular Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
  • 3 Curriculum in Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Computational Medicine Program, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
  • 4 Janelia Research Campus, The Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
  • 5 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Curriculum in Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Computational Medicine Program, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: [email protected].
  • 6 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Computational Medicine Program, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: [email protected].
PMID: 34637702 DOI: 10.1016/j.cell.2021.09.026
Abstract

We describe an approach to study the conformation of individual proteins during single particle tracking (SPT) in living cells. "Binder/tag" is based on incorporation of a 7-mer peptide (the tag) into a protein where its solvent exposure is controlled by protein conformation. Only upon exposure can the peptide specifically interact with a reporter protein (the binder). Thus, simple fluorescence localization reflects protein conformation. Through direct excitation of bright dyes, the trajectory and conformation of individual proteins can be followed. Simple protein engineering provides highly specific biosensors suitable for SPT and FRET. We describe tagSrc, tagFyn, tagSyk, tagFAK, and an orthogonal binder/tag pair. SPT showed slowly diffusing islands of activated Src within Src clusters and dynamics of activation in adhesions. Quantitative analysis and stochastic modeling revealed in vivo Src kinetics. The simplicity of binder/tag can provide access to diverse proteins.

Keywords

FRET; Src; adhesion; biosensor; cluster detection; conformation; living cell; single molecule; single particle tracking; stochastic modeling.

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