Abstract
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.
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References
Chiti, F., Stefani, M., Taddei, N., et al. (2003) Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature 424, 805–808.
Dobson, C. M. (2003) Protein folding and misfolding. Nature 426, 884–890
Evans, M. S., Clarke, T. F. IV, Clark, P. L. (2005) Conformations of co-translational folding intermediates. Protein Pept Lett 12, 189–195.
Minton, A. P. (2006) How can biochemical reactions within cells differ from those in test tubes? J Cell Sci 119, 2863–2869.
Pace, C. N., McGrath, T. (1980) Substrate stabilization of lysozyme to thermal and guanidine hydrochloride denaturation. J Biol Chem 255, 3862–3865.
Frand, A. R., Cuozzo, J. W., Kaiser, C. A. (2000) Pathways for protein disulphide bond formation. Trends Cell Biol 10, 203–210.
Ghaemmaghami, S., Oas, T. G. (2001) Quantitative protein stability measurement in vivo. Nat Struct Biol 8, 879–882.
Park, C., Marqusee, S. (2005) Pulse proteolysis: a simple method for quantitative determination of protein stability and ligand binding. Nat. Methods 2, 207–212.
Wigley, W. C., Stidham, R. D., Smith, N. M., et al. (2001) Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein. Nat Biotechnol 19, 131–136.
Philipps, B., Hennecke, J., Glockshuber, R. (2003) FRET-based in vivo screening for protein folding and increased protein stability. J Mol Biol 327, 239–249.
Maxwell, K. L., Mittermaier, A. K., Forman-Kay, J. D., et al. (1999) A simple in vivo assay for increased protein solubility. Protein Sci 8, 1908–1911.
Ignatova, Z., Krishnan, B., Bombardier, J. P., et al. (2007) From the test tube to the cell: Exploring the folding and aggregation of a β-clam protein. Biopolymers 88,157–163.
Ignatova, Z., Gierasch, L. M. (2004) Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling. Proc Natl Acad Sci USA 101, 523–528.
Clark, P. L., Weston, B. F., Gierasch, L. M. (1998) Probing the folding pathway of a beta-clam protein with single-tryptophan constructs. Fold. Des. 3, 401–412.
Griffin, B. A., Adams, S. R., Jones, J., Tsien, R. Y. (2000) Fluorescent labeling of recombinant proteins in living cells with FlAsH. Methods Enzymol. 327, 565–578.
Eyles, S. J., Gierasch, L. M. (2000) Multiple roles of prolyl residues in structure and folding. J Mol Biol 301, 737–747.
Ignatova, Z., Gierasch, L. M. (2005) Aggregation of a slow-folding mutant of a β-clam protein proceeds through a monomeric nucleus. Biochemistry 44, 7266–7274.
Racher, K. I., Culham, D. E., Wood, J. M. (2001) Requirements for osmosensing and osmotic activation of transporter ProP from Escherichia coli. Biochemistry 40, 7324–7333.
Ignatova, Z., Gierasch, L. M. (2006) Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant. Proc Natl Acad Sci USA 103, 13357–13361.
Ignatova, Z., Gierasch, L. M. (2007) Effects of osmolytes on protein folding and aggregation in cells. Meth Enzymol 428, 355–372.
Fersht, A. (1999) Structure and Mechanism in Protein Science, Freeman, New York, pp. 508–539.
Pace, C. N., Shirley, B.A., Thompson, J.A. (1987) In (Creighton, T.E., ed.), Protein structure: A practical approach, IRL, Oxford pp. 311–330.
Acknowledgments
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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Ignatova, Z., Gierasch, L.M. (2009). A Method for Direct Measurement of Protein Stability In Vivo. In: Shriver, J. (eds) Protein Structure, Stability, and Interactions. Methods in Molecular Biology, vol 490. Humana Press. https://doi.org/10.1007/978-1-59745-367-7_7
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DOI: https://doi.org/10.1007/978-1-59745-367-7_7
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