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Related Experiment Video

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Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
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Massively parallel, computationally guided design of a proenzyme.

Brahm J Yachnin1,2, Laura R Azouz1,2, Ralph E White3

  • 1Department of Chemistry & Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854.

Proceedings of the National Academy of Sciences of the United States of America
|April 4, 2022
PubMed
Summary

Scientists engineered a new method to create inactive proenzymes that can be activated by proteolysis. This breakthrough allows for precise control of enzyme activity in specific environments, minimizing side effects.

Keywords:
Rosettacarboxypeptidase G2proenzyme designprotein designzymogens

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Area of Science:

  • Protein engineering
  • Enzyme kinetics
  • Computational biology

Background:

  • Controlling protein activity in specific microenvironments is crucial for therapeutic applications.
  • Natural enzymes often exist as inactive zymogens, activated by proteolysis, but redesigning this feature is challenging.

Purpose of the Study:

  • To develop a computational design and screening approach for creating stimulus-responsive proenzymes.
  • To engineer carboxypeptidase G2 (CPG2) into a proenzyme for controlled therapeutic action.

Main Methods:

  • Utilized a massively parallel computational design, screening, and next-generation sequencing approach.
  • Employed carboxypeptidase G2 (CPG2) as a model system.
  • Performed kinetic, structural, and thermodynamic characterization of designed proenzymes.

Main Results:

  • Successfully designed CPG2 variants inhibited by ~80%, with activity fully restored by proteases.
  • Incorporating disulfide bonds increased inhibition to 98% but reduced protease-mediated activity restoration.
  • Disulfide-containing proenzymes showed significantly lower activity in cell culture compared to activated enzymes.

Conclusions:

  • The developed methodology enables the design of proproteins with precise spatial and temporal regulation.
  • This approach holds potential for developing cell type-specific therapeutics and controlling enzyme activity.
  • Insights into prodomain binding and inhibition mechanisms were gained through structural and thermodynamic studies.