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Science 3 August 2007:
Vol. 317. no. 5838, pp. 663 - 666
DOI: 10.1126/science.1139857
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Reports

Forced Unfolding of Proteins Within Cells

Colin P. Johnson,1*{dagger}{ddagger} Hsin-Yao Tang,2* Christine Carag,1{dagger}{ddagger} David W. Speicher,2{dagger} Dennis E. Discher1,2{dagger}{ddagger}

To identify cytoskeletal proteins that change conformation or assembly within stressed cells, in situ labeling of sterically shielded cysteines with fluorophores was analyzed by fluorescence imaging, quantitative mass spectrometry, and sequential two-dye labeling. Within red blood cells, shotgun labeling showed that shielded cysteines in the two isoforms of the cytoskeletal protein spectrin were increasingly labeled as a function of shear stress and time, indicative of forced unfolding of specific domains. Within mesenchymal stem cells—as a prototypical adherent cell—nonmuscle myosin IIA and vimentin are just two of the cytoskeletal proteins identified that show differential labeling in tensed versus drug-relaxed cells. Cysteine labeling of proteins within live cells can thus be used to fluorescently map out sites of molecular-scale deformation, and the results also suggest means to colocalize signaling events such as phosphorylation with forced unfolding.

1 Biophysical Engineering Lab, University of Pennsylvania, Philadelphia, PA 19104, USA.
2 Systems Biology Division—The Wistar Institute, Philadelphia, PA 19104, USA.

* These authors conducted experiments.

{dagger} These authors designed, refined, and analyzed experiments.

{ddagger} These authors modeled the data and wrote the paper.

To whom correspondence should be addressed. E-mail: discher{at}seas.upenn.edu

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