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Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues
Published on: July 14, 2015
Circular permutation prediction reveals a viable backbone disconnection for split proteins: an approach in
Yun-Tzai Lee1, Tz-Hsiang Su, Wei-Cheng Lo
1Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
Plos One
|September 1, 2012
Summary
This study shows that circular permutation sites can be used to create new split proteins, like split inteins. This method successfully generated a novel split intein with enhanced protein trans-splicing activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Split-protein systems are valuable for studying biomolecular interactions and reactions.
- Identifying reliable split sites for protein engineering remains a challenge.
Purpose of the Study:
- To demonstrate that valid circular permutation (CP) sites can serve as effective split sites for creating novel split proteins.
- To utilize CP prediction to identify permissive internal sites for split protein design.
- To engineer and characterize new split inteins with improved protein trans-splicing (PTS) activity.
Main Methods:
- Employed a computational circular permutation (CP) predictor to identify potential split sites in intein proteins.
- Created and screened intein circular permutants, focusing on those with minimal impact on protein folding.
- Assessed the folding thermodynamics and protein trans-splicing (PTS) activities of the engineered split inteins.
Main Results:
- Validated the feasibility of using CP sites as split sites, demonstrating favorable free energy for self-association despite altered folding thermodynamics.
- Identified stable, native-fold intein circular permutants.
- Discovered a novel functional split intein (N-terminal 36 residues + C-terminal fragment) exhibiting superior PTS activity compared to existing systems.
Conclusions:
- In silico CP prediction is a powerful strategy for designing functional split proteins, including split inteins.
- This approach facilitates the creation of split inteins with enhanced protein trans-splicing capabilities.
- The findings provide a new method for protein engineering and the development of split-protein tools.
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