A Method for Direct Measurement of Protein Stability In Vivo

  • Zoya Ignatova
  • Lila M. Gierasch
Part of the Methods in Molecular Biology book series (MIMB, volume 490)


The stability of proteins is tuned by evolution to enable them to perform their cellular functions for the success of an organism. Yet, most of the arsenal of biophysical techniques at our disposal to characterize the thermodynamic stability of proteins is limited to in vitro samples. We describe an approach that we have developed to observe a protein directly in a cell and to monitor a fluorescence signal that reports the unfolding transition of the protein, yielding quantitatively interpretable stability data in vivo. The method is based on incorporation of structurally nonperturbing, specific binding motifs for a bis-arsenical fluorescein derivative in sites that result in dye fluorescence differences between the folded and unfolded states of the protein under study. This fluorescence labeling approach makes possible the determination of thermodynamic stability by direct urea titration in Escherichia coli cells. The specific case study we describe was carried out on the predominantly β-sheet intracellular lipid-binding protein, cellular retinoic acid-binding protein (CRABP), expressed in E. coli.

Key words

Protein stability in-cell urea titration FlAsH labeling fluorescence microscopy CRABP 



We appreciate critical reading of the manuscript by Joanna Swain, Beena Krishnan, and Qinghua Wang. The authors gratefully acknowledge support from the National Institutes of Health (grants GM027616 and a 2006 NIH Director’s Pioneer Award to LMG), and DFG-project IG73/4-1 and the Heisenberg award IG73 1-1 (to ZI).


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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Zoya Ignatova
    • 1
  • Lila M. Gierasch
    • 2
  1. 1.Cellular Biochemistry, Max-Plank-Institute for BiochemistryGermany
  2. 2.Departments of Biochemistry and Molecular Biology and ChemistryUniversity of Massachusetts-AmherstAmherstUSA

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