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Engineering synthetic signaling proteins with ultrasensitive input/output control

Nature Biotechnology volume 25, pages 660–662 (2007)Cite this article

Abstract

Many signaling proteins are built from simple, modular components, yet display highly complex signal-processing behavior. Here we explore how modular domains can be used to build an ultrasensitive switch—a nonlinear input/output function that is central to many complex biological behaviors. By systematically altering the number and affinity of modular autoinhibitory interactions, we show that we can predictably convert a simple linear signaling protein into an ultrasensitive switch.

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Figure 1: Potential steps in the evolution or engineering of an ultrasensitive signaling node.
Figure 2: Design and synthesis of a modular ultrasensitive switch built from the N-WASP output domain and multiple SH3 modules.

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Acknowledgements

We thank J. Christopher Anderson, Adam Arkin, Noah Helman, Anton Vila-Sanjurjo, Brian Yeh and Brad Zamft for comments and discussion of this manuscript as well as members of the Lim laboratory for scientific advice. This work was supported by the National Institutes of Health and the National Science Foundation Synthetic Biology Engineering Research Center (SynBERC).

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Author notes

  1. John E Dueber

    Present address: Present address: Department of Synthetic Biology, California Institute of Quantitative Biomedical Research (QB3), University of California Berkeley, 717 Potter St., Berkeley, California 94720-3224, USA.,

Authors and Affiliations

  1. Program in Biological Sciences, University of California, San Francisco, 94158-2517, California, USA

    John E Dueber

  2. Graduate Group in Biophysics, University of California San Francisco, 600 16th Street, San Francisco, 94158-2517, California, USA

    Ethan A Mirsky

  3. Department of Cellular and Molecular Pharmacology, University of California San Francisco, 600 16th Street, San Francisco, 94158-2517, California, USA

    Wendell A Lim

  4. UCSF/UC Berkeley Nanomedicine Development Center, San Francisco, 94158-2517, California, USA

    Wendell A Lim

  5. NSF Synthetic Biology Engineering Research Center, 717 Potter St., Berkeley, 94720-3224, California, USA

    John E Dueber, Ethan A Mirsky & Wendell A Lim

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  1. John E Dueber
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  2. Ethan A Mirsky
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  3. Wendell A Lim
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Contributions

J.E.D. designed and conducted experiments depicted in this paper as well as constructing/purifying many of the synthetic switches described; prepared manuscript with W.A.L. E.A.M. constructed/purified many of the switches described, conducted experiments, and created model for switch parameter prediction; edited manuscript. W.A.L. made a major contribution to the conception of the general design of ultrasensitive switches and played a general mentorship role; prepared manuscript with J.E.D.

Corresponding author

Correspondence to Wendell A Lim.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Metric for relative activity of N-WASP switches based on half-time of actin polymerization. (PDF 553 kb)

Supplementary Fig. 2

Composition of switch linkers. (PDF 60 kb)

Supplementary Fig. 3

Intramolecular affinities must be properly tuned to generate a switch with targeted behavior: basal repression and sensitivity to external input. (PDF 40 kb)

Supplementary Fig. 4

Mutation of two peptide ligands in the five interaction switch (A.5.3b) reduces the Hill coefficient from ∼4 to 2.5. (PDF 37 kb)

Supplementary Fig. 5

Design and behavior of an AND-gate for three independent inputs. (PDF 139 kb)

Supplementary Table 1

Components used in switch construction and their properties (PDF 92 kb)

Supplementary Methods (PDF 485 kb)

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Dueber, J., Mirsky, E. & Lim, W. Engineering synthetic signaling proteins with ultrasensitive input/output control. Nat Biotechnol 25, 660–662 (2007). https://doi.org/10.1038/nbt1308

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  • DOI: https://doi.org/10.1038/nbt1308

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