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Cooperative Allosteric Transitions01:58

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Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
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Binding sites linkages can regulate a protein's function.  For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
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Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
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Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation
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Engineering proteins for allosteric control by light or ligands.

Onur Dagliyan1, Nikolay V Dokholyan2, Klaus M Hahn3

  • 1Department of Neurobiology, Harvard Medical School, Boston, MA, USA.

Nature Protocols
|May 12, 2019
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Summary
This summary is machine-generated.

This study presents a protocol for creating genetically encoded protein analogs controllable by rapamycin or blue light. These engineered proteins enable precise spatiotemporal control of protein activity in living cells for signaling circuit analysis.

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Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Biochemistry

Background:

  • Controlling protein activity in cells is crucial for understanding signaling pathways.
  • Engineered protein analogs offer a way to study protein function with minimal disruption.
  • Identifying suitable allosteric sites for domain insertion has been a significant challenge.

Purpose of the Study:

  • To develop a protocol for generating genetically encoded protein analogs with controllable allosteric responses.
  • To enable precise spatiotemporal regulation of protein activity in living cells.
  • To provide a method for interrogating signaling circuits using engineered proteins.

Main Methods:

  • Utilized computational methods (crystal structures, homology models) to identify allosteric sites for domain insertion.
  • Employed engineered rapamycin-responsive (uniRapR) or light-responsive (LOV2) domains.
  • Developed protocols for computational design, cloning, and in vitro/in vivo experimental validation.

Main Results:

  • Successfully generated and tested protein analogs regulated by rapamycin (irreversible activation) or blue light (reversible inactivation).
  • Demonstrated application to protein kinases, Rho GTPases, and Guanine Exchange Factors (GEFs), including Vav2 and Rac1.
  • Showcased high spatial and temporal resolution in controlling protein activity.

Conclusions:

  • The developed protocol provides a robust strategy for creating allosterically controlled protein analogs.
  • This approach facilitates the study of spatiotemporal dynamics in cellular signaling circuits.
  • The method is broadly applicable to various protein families and signaling pathways.