Optimizing Stability in Dynamic Small-Molecule Binding Proteins

Area of Science:

• Protein engineering
• Structural biology
• Biochemistry

Background:

• Protein function relies on conformational stability and energy barriers between states.
• Designing beneficial mutations is limited by unknown energy barrier details.
• Periplasmic binding proteins (PBPs) are dynamic, switching between open and closed states upon ligand binding.

Purpose of the Study:

• To develop a reliable protein stability design method by considering multiple conformational states.
• To engineer stabilized variants of periplasmic binding proteins (PBPs).
• To overcome limitations in protein design for dynamic proteins.

Main Methods:

• Hypothesized that designing mutations compatible with distinct equilibrium conformations enables reliable stability design.
• Focused on PBPs, constraining evolutionary and structural features of hinge and interface residues.
• Filtered incompatible mutations and designed stabilized variants from four different PBPs.

Main Results:

• Design based on a single conformation with evolutionary constraints was insufficient for wild-type binding affinity.
• Using mutations compatible with both conformations and structural constraints enhanced thermal stability.
• This approach mitigated trade-offs between stability and ligand binding affinity.

Conclusions:

• A straightforward method for one-shot stabilization of dynamic proteins was demonstrated.
• This method provides robust starting points for thermostable and responsive biosensors.
• Considering multiple conformations is key for successful protein stabilization design.