Loop size optimization induces a strong thermal stabilization of the thioredoxin fold

Area of Science:

• Structural biology
• Protein chemistry
• Biophysics

Background:

• Protein thermostability is crucial in structural biology.
• Proteins from thermophilic organisms often follow specific topological rules.
• Baker and coworkers previously identified fundamental rules of protein topology.

Purpose of the Study:

• To investigate if modifying natural proteins based on topological rules can enhance their stability.
• To design and characterize novel variants of Escherichia coli thioredoxin (EcTrx) for improved structural stability.

Main Methods:

• Designed chimeric variants of EcTrx by replacing its loops with those from thermophilic Trxs (Sulfolobus solfataricus and Sulfolobus tokodaii).
• Employed a range of biophysical and structural techniques for characterization.
• Determined the crystallographic structure of a stabilized variant.

Main Results:

• Insertion of loops from thermophilic Trxs into EcTrx induced remarkable protein stabilization (≥10 °C).
• Loop sequences showed no significant similarity, indicating topological rules, not sequence homology, drove stabilization.
• Crystallographic data confirmed that loop size optimization is the primary factor in stabilization.

Conclusions:

• Ad hoc modification of protein loop size, guided by topological rules, is an effective strategy for protein stabilization.
• This study provides a proof of concept for stabilizing natural proteins using topological rules.
• A novel protocol for protein stabilization applicable to other proteins is proposed.