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Allosteric Regulation

Allosteric Regulation

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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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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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.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...

Ligand Binding and Linkage

Ligand Binding and Linkage

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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Related Experiment Video

Updated: May 13, 2025

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

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Published on: January 30, 2018

Domain Coupling in Allosteric Regulation of SthK Measured Using Time-Resolved Transition Metal Ion FRET.

Pierce Eggan¹, Sharona E Gordon¹, William N Zagotta¹

¹Department of Neurobiology and Biophysics, University of Washington, Seattle, WA 98195.

Biorxiv : the Preprint Server for Biology

|April 16, 2025

Summary

Cyclic nucleotide-binding domain (CNBD) ion channels are regulated by cyclic nucleotide binding. Using time-resolved tmFRET, we found that transmembrane domains make the activating conformational change in SthK CNBD more favorable.

Keywords:

CNG channel allostery conformational dynamics

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Last Updated: May 13, 2025

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Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Published on: March 14, 2019

Area of Science:

Biophysics
Structural Biology
Molecular Biology

Background:

Cyclic nucleotide-binding domain (CNBD) ion channels are crucial for cellular signaling and excitability.
Activation of these channels is mediated by cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP) binding.
The allosteric mechanisms and energetics governing conformational changes in CNBD channels are not fully understood.

Purpose of the Study:

To investigate the conformational dynamics and energetics of the SthK CNBD using time-resolved transition metal ion Förster resonance energy transfer (tmFRET).
To elucidate the allosteric mechanisms underlying ligand-mediated activation in CNBD channels.
To quantify the energetics of domain coupling within the SthK channel.

Main Methods:

Application of time-resolved tmFRET with a noncanonical amino acid (Acd) as a FRET donor.
Utilizing a metal-chelator conjugate on cysteine as a FRET acceptor.
Employing time-correlated single photon counting (TCSPC) to measure FRET and determine donor-acceptor distance distributions.

Main Results:

Conformational dynamics and energetics of SthK CNBD were analyzed in both soluble (SthK_Cterm) and full-length (SthK_Full) forms.
Donor-acceptor distance distributions were obtained in the absence and presence of cAMP.
The presence of transmembrane domains in SthK_Full was found to stabilize the activated conformation of the CNBD, making the activating conformational change more energetically favorable.

Conclusions:

Time-resolved tmFRET is a powerful technique for characterizing the structural and energetic landscapes of allosteric proteins.
The study provides insights into the ligand-mediated activation mechanism of CNBD channels.
Transmembrane domains play a significant role in modulating the allosteric response of the CNBD.