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Related Concept Videos

Ligand Binding Sites02:40

Ligand Binding Sites

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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.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
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Ligand Binding and Linkage00:49

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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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Related Experiment Video

Updated: Jun 4, 2025

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy
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Structural Basis of High-Precision Protein Ligation and Its Application.

Kelvin Han Chung Chong1,2, Lichao Liu3, Rae Chua2,4

  • 1School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 636921, Singapore.

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|January 2, 2025
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Summary

Researchers engineered Connectase, a novel enzyme for precise protein ligation, overcoming limitations of existing methods. Structural insights enabled modifications for high-precision protein conjugation in complex biological settings.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Engineering

Background:

  • Enzyme-catalyzed protein modifications offer superior precision and compatibility compared to chemical methods.
  • Existing ligases like sortase A and OaAEP1 have limitations in target specificity and application in complex biological environments.
  • Connectase, a repurposed protease, showed promise for protein ligation but lacked processivity and structural data for optimization.

Purpose of the Study:

  • To elucidate the structural basis of MmConnectase (MmCET) activity through X-ray crystallography.
  • To engineer Connectase for enhanced precision and processivity in protein ligation.
  • To enable high-precision protein conjugation in challenging biological milieu.

Main Methods:

  • X-ray crystallography of MmConnectase in apo and substrate-bound states.
  • Comparative structural analysis with MjCET to identify key functional differences.
  • Site-directed mutagenesis targeting N-terminal substrate recognition motifs.
  • Single-molecule protein unfolding experiments to assess ligation efficiency and processivity.

Main Results:

  • Determined the X-ray crystal structures of MmConnectase, revealing the structural basis for its ligation activity.
  • Identified key structural features distinguishing MmConnectase from its inactive paralogue, MjCET.
  • Proposed and validated modifications to suppress protease activity and enhance ligation precision.
  • Demonstrated stepwise tandem ligations using optimized Connectase and OaAEP1(C247A) for polymer formation.

Conclusions:

  • Structural insights into MmConnectase facilitate its engineering for advanced protein ligation.
  • Modified Connectase enables high-precision protein conjugation in complex biological environments like cell cultures.
  • Optimized Connectase demonstrates enhanced processivity for creating defined protein polymers.