Heterochiral Knottin Protein: Folding and Solution Structure.

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Biological macromolecules predominantly exhibit homochirality.
• Heterochiral proteins, containing both d- and l-amino acids, are rare in nature.
• Understanding their structure and function is crucial for expanding protein design possibilities.

Purpose of the Study:

• To design and synthesize heterochiral proteins with opposite chirality loops.
• To investigate the folding, stability, and structural properties of these novel proteins.
• To explore the potential of incorporating both d- and l-amino acids in protein architectures.

Main Methods:

• Chemical synthesis of heterochiral protein constructs.
• Utilizing Ecballium elaterium trypsin inhibitor II as a scaffold.
• Employing solution nuclear magnetic resonance (NMR) spectroscopy for structural characterization.
• Performing steered molecular dynamics simulations to assess folding energy.
• Evaluating resistance to proteolysis compared to homochiral proteins.

Main Results:

• Selective beta-alanine substitution facilitated efficient folding of heterochiral constructs.
• NMR spectroscopy confirmed a homogeneous global fold in the synthesized heterochiral protein.
• Molecular dynamics simulations indicated reduced folding free energy with beta-alanine.
• Heterochiral proteins demonstrated enhanced resistance to proteolysis compared to homochiral counterparts.

Conclusions:

• Beta-alanine substitution is key for successful heterochiral protein folding.
• Heterochiral proteins exhibit stable structures and increased proteolytic resistance.
• This study provides a foundation for designing novel protein architectures with mixed chirality.