Optogenetic control of protein binding using light-switchable nanobodies

Gil, Agnieszka A.; Zhao, Evan M.; Wilson, Maxwell Z.; Goglia, Alexander G.; Carrasco-López, César; Avalos, José L.; Toettcher, Jared E.

doi:10.1101/739201

Cited by 19 publications

(31 citation statements)

References 43 publications

(62 reference statements)

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“…To build our chimeras, we used a truncated version of AsLOV2, which induces light-dependent conformational changes in engineered nanobody chimeras more efficiently than its full-length counterpart 45 . Our strategy was to insert the shortened AsLOV2 domain in all seven structurally-conserved, solvent-exposed loops of HA4 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, the light responsiveness of OptoMB is effective to control binding to proteins fused to SH2 at either their N- or C-terminus. OptoMBs, along with the light-dependent nanobodies (OptoNBs) we describe in an accompanying study 45 , belong to a new class of light-dependent protein binders we call OptoBinders (OptoBNDRs), which offer promising new in vivo and in vitro applications.…”

Section: Discussionmentioning

confidence: 99%

“…A stop codon was added before the in-frame (C-terminus) 6x-histidine tag of pCri-7b. The AsLOV2 domain (residues 408-543) was either amplified by PCR from previous constructs 45 (wt AsLOV2), or synthesized as IDT gBlocks (AsLOV2 mutants). To insert AsLOV2 into the monobody, the backbone from the initial construct containing HA4 (EZ-L663) was PCR amplified, using Takara Hifi PCR premix, starting from the insertion positions (Supplementary Table 1) that were selected (and adding homology arms to AsLOV2).…”

Section: Methodsmentioning

confidence: 99%

“…To build the structural model of the HA4-AsLOV2 chimera (OptoMB) interacting with the SH2 domain (Fig. 1e), a shortened version 45 (residues 408-543) of the AsLOV2 domain (PDB ID: 2V1A) 64 was manually inserted to residues S58 and S59 of the monobody HA4, using the program Coot 65 . The crystal structure of HA4 in complex with the SH2 domain (PDB ID: 3k2m) 46 was used as template.…”

Section: Methodsmentioning

confidence: 99%

“…We harness the ~300-fold change in binding affinity between lit and dark states to implement light-based protein purification 44 using an OptoMB resin in what we call “light-controlled affinity chromatography” (LCAC). This work, and an accompanying study reporting light-dependent nanobodies or OptoNanobodies (OptoNBs) 45 , represent the first demonstrations of protein binders with high affinity and selectivity, engineered with light-control of their binding activity.…”

Section: Introductionmentioning

confidence: 99%

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Light-responsive monobodies for dynamic control of customizable protein binding

Carrasco-López

Zhao

Gil

et al. 2020

Preprint

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Customizable, high affinity protein-protein interactions, such as those mediated by antibodies and antibody-like molecules, are invaluable to basic and applied research and have become pillars for modern therapeutics. The ability to reversibly control the binding activity of these proteins to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, a light-controlled monobody (OptoMB) that works in vitro and in vivo, whose affinity for its SH2-domain target exhibits a 300-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. This OptoMB belongs to a new class of light-sensitive protein binders we call OptoBinders (OptoBNDRs) which, by virtue of their ability to be designed to bind any protein of interest, have the potential to find new powerful applications as light-switchable binders of untagged proteins with high affinity and selectivity, and with the temporal and spatial precision afforded by light.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Light-responsive monobodies for dynamic control of customizable protein binding

Carrasco-López

Zhao

Gil

et al. 2020

Preprint

Self Cite

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Enlightening Allostery: Designing Switchable Proteins by Photoreceptor Fusion

Mathony

Niopek

2020

Advanced Biology

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upon photoexcitation and often reversible at similar time-scales upon withdrawal of the light trigger. [1][2][3][4] Apart from this temporal precision, light can also be supplied in a spatially confined way with single-cell or subcellular accuracy, for example by using lasers or blinds. [5][6][7] Together, these favorable characteristics render optogenetics a highly powerful method for perturbing and studying dynamic processes in living cells and explain the rapid growth of this scientific field over the past 15 years.Generally, optogenetic tools can either be categorized by the nature of the photoreceptors they employ or by their undelaying working principle. [8] Focusing on the latter classification, one can differentiate between systems that are regulated via inducible association/dissociation reactions and often involve multiple component versus single-component tools functioning via inducible allostery. [8] Association-based tools make use of photoreceptors that bind to one another or to a secondary binding partner upon light exposure. [9] The red/far-red light-responsive PhyB/PIF or blue light-responsive CRY2/CIB1 heterodimerization systems are typical examples for association-dependent optogenetic tools. One application of such optogenetic heterodimerizers is the control of protein subcellular localization. [10] To this end, one binding partner is targeted to a specific subcellular structure or location (e.g., the plasma membrane, nucleus, mitochondria), while the second partner is corecruited to this structure upon dimerization. The same principle can also be employed to reconstitute split-proteins in a light-dependent manner. [9] Optogenetic tools that function via inducible allostery, in contrast, are usually single-component systems. They consist of an engineered chimera between a photoreceptor domain and an effector protein and their light-switching behavior is mediated by steric or allosteric interactions between the two. [8,11] A particular advantage of single-component optogenetic tools as compared to dimerization-based tools is their compactness. They are small in size, since only the photosensory domain is fused to the effector domain/protein of choice and no additional proteins need to be coexpressed. Moreover, allosteric tools do not rely on the diffusion-based association of two components, the kinetics of which depends on the protein concentrations.Optogenetic switches that rely on inducible allostery have mainly been engineered on the basis of light-oxygen-voltage (LOV) domains [11] and phytochromes. [12][13][14] LOV domains belong to the Per-ARNT-Sim or PAS (period circadian protein-aryl Optogenetics harnesses natural photoreceptors to non-invasively control selected processes in cells with previously unmet spatiotemporal precision. Linking the activity of a protein of choice to the conformational state of a photosensor domain through allosteric coupling represents a powerful method for engineering light-responsive proteins. It enables the design of compact and highly potent single-componen...

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O‐GlcNAc Engineering on a Target Protein in Cells with Nanobody‐OGT and Nanobody‐splitOGA

Ramirez

Woo

2021

Current Protocols

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The monosaccharide O-linked N-acetyl glucosamine (O-GlcNAc) is an essential and dynamic post-translational modification (PTM) that decorates thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a target protein is crucial yet challenging to perform in cells. We recently reported a pair of methods to selectively install or remove O-GlcNAc on a target protein in cells using an engineered O-GlcNAc transferase (OGT) or split O-GlcNAcase (OGA) fused to a nanobody. Target protein O-GlcNAcylation and de-O-GlcNAcylation complements methods to interrogate the role of O-GlcNAc on a global scale or at individual glycosites. Herein, we describe a protocol for utilizing the nanobody-OGT and nanobody-splitOGA systems to screen for O-GlcNAc functionality on a target protein. We additionally include associated protocols for the detection of O-GlcNAc and cloning procedures to adapt the method for the user's target protein of interest.

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Optogenetic control of protein binding using light-switchable nanobodies

Cited by 19 publications

References 43 publications

Light-responsive monobodies for dynamic control of customizable protein binding

Light-responsive monobodies for dynamic control of customizable protein binding

Enlightening Allostery: Designing Switchable Proteins by Photoreceptor Fusion

O‐GlcNAc Engineering on a Target Protein in Cells with Nanobody‐OGT and Nanobody‐splitOGA

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