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Active barnase variants with completely random hydrophobic cores
D D Axe1, N W Foster, A R Fersht
1Medical Research Council Unit for Protein Function and Design, Department of Chemistry, University of Cambridge, United Kingdom.
Summary
Hydrophobicity alone is nearly sufficient for creating functional protein cores. This finding simplifies developing novel enzymes through design or evolution by lowering initial hurdles.
Area of Science:
- Protein Engineering
- Enzyme Design
- Biochemistry
Background:
- Natural proteins feature highly ordered hydrophobic cores essential for enzymatic function.
- Developing novel enzymes is challenging if native-like core packing is strictly required.
Purpose of the Study:
- To determine the minimum requirements for a hydrophobic core to maintain enzymatic activity.
- To investigate the role of hydrophobicity versus specific residue packing in protein core function.
Main Methods:
- Mutagenesis of barnase ribonuclease, randomly replacing 12 of 13 core residues with hydrophobic alternatives.
- Utilizing a sensitive biological screen to assess enzymatic activity of mutants in vivo.
- Further substitutions at the 13th core position to explore a completely randomized hydrophobic core.
Main Results:
- A high proportion (23%) of randomly mutated barnase retained enzymatic activity.
- Completely randomized hydrophobic cores also supported enzyme function.
- Several active mutants lacked any wild-type core residues, indicating sequence flexibility.
Conclusions:
- Hydrophobicity is a nearly sufficient criterion for constructing a functional protein core.
- Initial enzyme evolution or design may require less stringent core constraints than previously thought.
- Simplifying core design to focus on hydrophobicity can facilitate the development of novel enzymes.