A mixed-alpha,beta miniprotein stereochemically reprogrammed to high-binding affinity for acetylcholine.

Area of Science:

• Protein engineering and de novo design
• Stereochemistry and molecular recognition
• Biophysical chemistry and molecular modeling

Background:

• Protein structure is primarily limited to L-amino acid configurations, while function is expanded by diverse side chains.
• Combining stereochemical diversity with chemical diversity offers new avenues for de novo protein design.
• Current methods allow for the creation of novel molecular shapes and functions through precise amino acid manipulation.

Purpose of the Study:

• To explore de novo protein design by incorporating both L and D amino acids.
• To reprogram canonical protein folds into novel shapes like bracelets, boats, and canoes.
• To demonstrate the chemical customization of molecular function, exemplified by creating an acetylcholine receptor.

Main Methods:

• Stereochemical reprogramming of L-amino acid residues to D-amino acid residues in protein structures.
• Utilizing techniques such as Circular Dichroism (CD), fluorescence, Nuclear Magnetic Resonance (NMR), Differential Scanning Calorimetry (DSC), Isothermal Titration Calorimetry (ITC), Molecular Dynamics (MD), and molecular docking.
• Designing a 15-residue mixed-alpha, beta miniprotein with specific stereochemical and chemical properties.

Main Results:

• Canonical all-beta folds were successfully reprogrammed into bracelet, boat, and canoe shapes.
• The 'boat' and 'canoe' structures demonstrated receptor-like pockets and metal-ion receptor capabilities, respectively.
• A 15-residue mixed-L, D peptide was engineered as a small, simple acetylcholine receptor with submicromolar affinity, mimicking natural and artificial acetylcholine receptor sites through cation-pi interactions.

Conclusions:

• De novo protein design incorporating stereochemical diversity (L and D amino acids) allows for precise control over molecular shape and function.
• This approach enables the creation of novel protein architectures with tailored chemical functionalities.
• The developed miniprotein serves as a highly efficient and simplified acetylcholine receptor, showcasing the potential of stereochemically programmed peptides.