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Active site center redesign increases protein stability preserving catalysis in thioredoxin
Maria Luisa Romero1,2,3, Hector Garcia Seisdedos1,4,5, Beatriz Ibarra-Molero1,5
1Departamento de Química Física, Universidad de Granada, Granada.
Protein Science : a Publication of the Protein Society
|September 17, 2024
Summary
Researchers enhanced protein stability by engineering thioredoxin. A single surface mutation restored catalytic activity and achieved high thermal stability (137°C) without compromising function.
Area of Science:
- Protein Engineering
- Biochemistry
- Structural Biology
Background:
- Protein stabilization is crucial for applications, with core hydrophobicity optimization being a common strategy.
- Buried catalytic residues essential for function often limit stability enhancement strategies.
- Thioredoxin's active site presents a challenge due to buried charged residues impacting stability and function.
Purpose of the Study:
- To enhance thioredoxin stability by modifying buried charged residues.
- To restore catalytic activity after initial stability modifications.
- To engineer a hyperstable thioredoxin variant with retained function.
Main Methods:
- Charged-to-hydrophobic substitution in the thioredoxin active site.
- Combinatorial library design targeting surface residues adjacent to the active site.
- Partial least squares regression for predicting mutational effects on stability and activity.
Main Results:
- Initial modification abolished catalytic activity but significantly increased stability.
- Over 50% of surface variants restored partial catalytic activity.
- A single surface point mutation fully restored activity without compromising thermostability, yielding a variant stable at 137°C.
Conclusions:
- Surface residue engineering can restore function to modified proteins without stability loss.
- Achieved unprecedented thermal stability in a naturally folded protein.
- The engineered hyperstable thioredoxin retains catalytic activity in vitro and in vivo.
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