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Cooperative Allosteric Transitions

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Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
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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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Protein Complexes with Interchangeable Parts01:57

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Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
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Riboswitches are non-coding mRNA domains that regulate the transcription and translation of downstream genes without the help of proteins. Riboswitches bind directly to a metabolite and can form unique stem-loop or hairpin structures in response to the amount of the metabolite present. They have two distinct regions – a metabolite-binding aptamer and an expression platform.
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Allosteric regulation of enzymes occurs when the binding of an effector molecule to a site that is different from the active site causes a change in the enzymatic activity. This alternate site is called an allosteric site, and an enzyme can contain more than one of these sites. Allosteric regulation can either be positive or negative, resulting in an increase or decrease in enzyme activity. Most enzymes that display allosteric regulation are metabolic enzymes involved in the degradation or...
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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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Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library
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Small-Molecule Modulated Affinity-Tunable Semisynthetic Protein Switches.

Chien-Chi Wu1, Shao-Jie Huang1, Tsung-Yu Fu1

  • 1Department of Chemistry, National Tsing Hua University, 101 Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan, Republic of China.

ACS Sensors
|September 9, 2022
PubMed
Summary

This study introduces a novel protein switch that uses target binding to control streptavidin-biotin interactions for signal generation. This engineered biosensor enables selective detection of small molecules on cell surfaces.

Keywords:
affinity-tunablebiotinsemisynthetic protein switchsmall-molecule detectionstreptavidin

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Area of Science:

  • Biotechnology
  • Molecular Engineering
  • Biosensing

Background:

  • Engineered protein switches are crucial for cell-based sensors and point-of-care diagnostics.
  • Current protein switches rely on optical property changes or enzyme activation for signal transduction.
  • A need exists for new switching mechanisms with tunable affinity and modularity.

Purpose of the Study:

  • To develop a new affinity-tunable protein switch strategy.
  • To enable signal generation through target-induced activation of streptavidin-biotin interaction.
  • To demonstrate selective detection of small molecules on cell surfaces.

Main Methods:

  • Engineered a protein switch where small-molecule binding modulates biotin availability.
  • Utilized a closed conformation with steric hindrance to block streptavidin binding in the absence of a target.
  • Developed an open conformation upon target binding, exposing biotin for streptavidin interaction and signal generation.
  • Employed fluorescent dyes and various detection techniques for readout.

Main Results:

  • The novel protein switch strategy successfully generated a signal upon target binding.
  • Target binding removed steric hindrance, allowing streptavidin-biotin interaction and strong fluorescent signals.
  • Selective detection of sulfonamides, methotrexate, and trimethoprim was achieved.
  • The sensing concept was validated on native and genetically engineered cells.

Conclusions:

  • The developed affinity-tunable protein switch offers a new modular sensing strategy.
  • This approach enables sensitive and selective detection of small molecules via induced streptavidin-biotin interaction.
  • The platform holds potential for diverse applications in cell-based sensing and diagnostics.