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Genetic selection designed to stabilize proteins uncovers a chaperone called Spy

Nature Structural & Molecular Biology volume 18, pages 262–269 (2011)Cite this article

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Abstract

To optimize the in vivo folding of proteins, we linked protein stability to antibiotic resistance, thereby forcing bacteria to effectively fold and stabilize proteins. When we challenged Escherichia coli to stabilize a very unstable periplasmic protein, it massively overproduced a periplasmic protein called Spy, which increases the steady-state levels of a set of unstable protein mutants up to 700-fold. In vitro studies demonstrate that the Spy protein is an effective ATP-independent chaperone that suppresses protein aggregation and aids protein refolding. Our strategy opens up new routes for chaperone discovery and the custom tailoring of the in vivo folding environment. Spy forms thin, apparently flexible cradle-shaped dimers. The structure of Spy is unlike that of any previously solved chaperone, making it the prototypical member of a new class of small chaperones that facilitate protein refolding in the absence of energy cofactors.

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Figure 1: A dual fusion selection for enhancing in vivo protein stability.
Figure 2: Overall experimental scheme.
Figure 3: Im7 and Spy are abundant in the periplasm of EMS strains.
Figure 4: Spy has chaperone activity.
Figure 5: Spy protects DsbB, aldolase and alkaline phosphatase from tannic acid–induced activity loss.
Figure 6: Crystal structure of the Spy dimer shown in three orientations rotated by 90° along the vertical axis.
Figure 7: Spy binds the disordered model substrate protein casein and the in vivo substrate protein Im7-L53A I54A.

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Acknowledgements

We thank G. Morgan and S. Radford for communicating results before publication and for the Im7-L53A I54A protein, T. Franzmann for conducting the ultracentrifugation experiments, D. Reichmann, C. Cremers and A. Malik for advice, M. Lei and Y. Chen for providing vector and reagents for Spy purification and C. Munger for initial purification and crystallization of Spy. Protein identification was done by the Protein Structure Facility at the University of Michigan. Diffraction data were collected at the Canadian Macromolecular Crystallography Facility-1 beamline at the Canadian Light Source. We would like to thank S. Labiuk for data collection. The Howard Hughes Medical Institute funded this work (to J.C.A.B). M.C. acknowledges financial support from Canadian Institutes of Health Research grant GSP-48370.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Shu Quan, Philipp Koldewey, Tim Tapley, Nadine Kirsch, Jennifer Pfizenmaier, Stephan Hofmann, Linda Foit, Guoping Ren & James C A Bardwell

  2. Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA

    Shu Quan, Philipp Koldewey, Tim Tapley, Nadine Kirsch, Jennifer Pfizenmaier, Stephan Hofmann, Linda Foit, Guoping Ren, Ursula Jakob & James C A Bardwell

  3. Department of Biochemistry, McGill University, Montreal, Quebec, Canada

    Karen M Ruane, Rong Shi & Miroslaw Cygler

  4. Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA

    Zhaohui Xu

  5. Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, USA

    Zhaohui Xu

  6. Biotechnology Research Institute, National Research Council, Montreal, Quebec, Canada

    Miroslaw Cygler

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Contributions

J.C.A.B. designed the study and wrote the manuscript, with contributions from M.C. and S.Q. S.Q., P.K., T.T., N.K., K.M.R., R.S., J.P., S.H. and G.R. conducted the experiments and collected and analyzed the data. J.C.A.B., Z.X. and M.C. further analyzed the data. L.F. and U.J. provided technical support and conceptual advice.

Corresponding author

Correspondence to James C A Bardwell.

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Quan, S., Koldewey, P., Tapley, T. et al. Genetic selection designed to stabilize proteins uncovers a chaperone called Spy. Nat Struct Mol Biol 18, 262–269 (2011). https://doi.org/10.1038/nsmb.2016

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