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Related Experiment Videos

Modular enzyme design: regulation by mutually exclusive protein folding.

Jeung-Hoi Ha1, James S Butler, Diana M Mitrea

  • 1Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, 750 E. Adams St, Syracuse, NY 13210, USA.

Journal of Molecular Biology
|February 18, 2006
PubMed
Summary

This study presents a novel regulatory mechanism where enzyme activity is controlled by ligand binding. A fused protein switches between active and inactive states, enabling new sensor capabilities.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Enzyme activity regulation is crucial for cellular processes.
  • Controlling enzyme function through external stimuli remains a challenge.
  • Modular protein design offers a pathway to engineer novel functions.

Purpose of the Study:

  • To introduce a regulatory mechanism for enzyme catalytic activity controlled by ligand binding.
  • To engineer a chimeric protein (Barnase-GCN4) that switches function based on DNA binding.
  • To demonstrate a modular approach for creating enzymes with sensing capabilities.

Main Methods:

  • Fusion of a catalytic enzyme (barnase) with a ligand-binding polypeptide (GCN4).
  • Exploitation of topological constraints and thermodynamic principles to control protein folding.

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  • Characterization of the chimeric protein's activity and structural changes upon ligand binding.

    • Main Results:

    • The Barnase-GCN4 chimera functions as a "natively unfolded" protein.
    • Ligand (DNA) binding induces folding of GCN4 and unfolding/inactivation of barnase.
    • Key characteristics of parent proteins (barnase catalytic efficiency, GCN4 DNA binding) are retained.
    • The system exhibits a "thermodynamic tug-of-war" between the two domains.

    Conclusions:

    • Barnase-GCN4 demonstrates a viable strategy for ligand-inducible enzyme regulation.
    • This modular approach allows for the assembly of enzymes with novel sensing capabilities.
    • The engineered protein retains parent domain functionalities, highlighting the modular design's effectiveness.