Evolution-based protein engineering: functional switching between transthyretins and 5-hydroxyisourate hydrolases.

Area of Science:

• Biochemistry
• Evolutionary Biology
• Protein Engineering

Background:

• Transthyretin (TTR) is a vertebrate transport protein crucial for thyroid hormone distribution.
• TTR evolved from the gene encoding 5-hydroxyisourate hydrolase (HIUase), an enzyme involved in uric acid metabolism.
• HIUase is widespread, while TTR is vertebrate-specific; both form homotetramers with similar structures.

Purpose of the Study:

• To investigate the potential for interconverting HIUase and TTR functions through computational protein engineering.
• To explore an evolution-based approach for modifying protein function by leveraging conserved residues.

Main Methods:

• Engineered novel protein sequences by substituting correlated locally conserved positions between HIUase and TTR representatives.
• Utilized computational modeling for refining, validating, and analyzing structural properties (cavity volume, geometry, aggregation propensity, electrostatics).
• Performed molecular dynamics simulations to assess the stability of engineered mutants in bound and unbound states.

Main Results:

• Computational analysis revealed differences in cavity volumes and geometries between engineered proteins and their ligands.
• Molecular dynamics simulations confirmed the stability of bound-state mutant complexes.
• Enzymatic assays demonstrated that the engineered mutants possessed active, novel enzymatic functions.

Conclusions:

• The evolution-based protein engineering strategy effectively created functional enzymes with altered properties.
• This approach allows for residue-specific modifications, highlighting conserved residues in evolutionarily related proteins like HIUase and TTR.
• The study provides a framework for designing proteins with tailored functions based on evolutionary relationships.