Engineering proteins for allosteric control by light or ligands

Onur Dagliyan, Nikolay Dokholyan, Klaus M. Hahn

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Control of protein activity in living cells can reveal the role of spatiotemporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective in the interrogation of protein signaling, as they can replace endogenous proteins with minimal perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces an allosteric response comparable to the activity of native proteins. Here, we describe a detailed protocol to generate genetically encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, as well as experimental procedures to produce and test these analogs in vitro and in mammalian cell lines. We describe computational methods, based on crystal structures or homology models, to identify effective sites for insertion of either an engineered rapamycin-responsive (uniRapR) domain or the light-responsive light–oxygen–voltage 2 (LOV2) domain. The inserted domains allosterically regulate the active site, responding to rapamycin with irreversible activation, or to light with reversible inactivation at higher spatial and temporal resolution. These strategies have been successfully applied to catalytic domains of protein kinases, Rho family GTPases, and guanine exchange factors (GEFs), as well as the binding domain of a GEF Vav2. Computational tasks can be completed within a few hours, followed by 1–2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, GEF Vav2, and Rho GTPase Rac1 as examples.

Original languageEnglish (US)
Pages (from-to)1863-1883
Number of pages21
JournalNature Protocols
Volume14
Issue number6
DOIs
StatePublished - Jun 1 2019

Fingerprint

Protein Engineering
Ligands
Light
Guanine
Sirolimus
Proteins
rho GTP-Binding Proteins
Catalytic Domain
Cells
Allosteric Site
src-Family Kinases
Cloning
GTP Phosphohydrolases
Computational methods
Protein Kinases
Organism Cloning
Research Design
Crystal structure
Chemical activation
Cell Line

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Dagliyan, Onur ; Dokholyan, Nikolay ; Hahn, Klaus M. / Engineering proteins for allosteric control by light or ligands. In: Nature Protocols. 2019 ; Vol. 14, No. 6. pp. 1863-1883.
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Engineering proteins for allosteric control by light or ligands. / Dagliyan, Onur; Dokholyan, Nikolay; Hahn, Klaus M.

In: Nature Protocols, Vol. 14, No. 6, 01.06.2019, p. 1863-1883.

Research output: Contribution to journalArticle

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