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Hypocrea jecorina cellobiohydrolase I stabilizing mutations identified using noncontiguous recombination
Matthew A Smith1, Claire N Bedbrook, Timothy Wu
1Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States.
ACS Synthetic Biology
|May 22, 2013
Summary
Noncontiguous recombination (NCR) efficiently creates novel chimeric proteins by shuffling structural blocks. This method rapidly identifies stabilizing mutations, enhancing protein stability with minimal experiments.
Area of Science:
- Protein engineering
- Biochemistry
- Structural biology
Background:
- Noncontiguous recombination (NCR) enables the creation of chimeric proteins by swapping structural units from homologous proteins.
- These structural units, or "blocks," are encoded by non-contiguous sequence elements within the polypeptide chain.
Purpose of the Study:
- To design and analyze a library of chimeric enzymes using NCR.
- To investigate the additive contribution of structural blocks to protein stability.
- To identify stabilizing amino acid substitutions in cellobiohydrolase I.
Main Methods:
- Designed a library of 531,438 chimeric enzymes by shuffling structural blocks from Hypocrea jecorina cellobiohydrolase I (Cel7A) and its thermostable homologues.
- Constructed and analyzed a subset of 35 chimeras to assess stability contributions.
- Identified single amino acid substitutions within stabilizing blocks.
Main Results:
- Structural blocks contribute additively to the stability of chimeric enzymes.
- Six single amino acid substitutions were identified, each increasing the stability of H. jecorina cellobiohydrolase I by 1-3 °C.
- The analysis required a small number of measurements, highlighting NCR's efficiency.
Conclusions:
- Noncontiguous recombination is an effective strategy for designing chimeric proteins with enhanced stability.
- NCR facilitates the efficient identification of single amino acid mutations that significantly improve protein thermostability.
- This approach offers a powerful tool for protein engineering and directed evolution.
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