Peptide Folding and Binding Probed by Systematic Non-canonical Mutagenesis

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Protein and peptide folding upon binding are crucial for molecular recognition.
• Mutagenesis is a key tool for dissecting multi-step binding and folding processes.
• Traditional mutagenesis is limited in scale for comprehensive analysis.

Purpose of the Study:

• To explore the utility of deep mutational scanning (DMS) for studying protein-peptide interactions.
• To investigate the impact of non-canonical amino acids on peptide structure-activity relationships.
• To guide the development of de novo cyclic peptides as therapeutic agents and chemical probes.

Main Methods:

• Utilized deep mutational scanning to analyze thousands of mutations in parallel.
• Incorporated mutations to non-canonical amino acids to expand structure-activity relationship analysis.
• Applied biophysical methods to assess the behavior of selected mutants.

Main Results:

• DMS enables parallel examination of numerous mutations, significantly increasing throughput.
• Inclusion of non-canonical amino acids provides unprecedented depth in understanding peptide structure-activity.
• The approach effectively separates and assesses interactions and conformational changes during folding and binding.

Conclusions:

• Deep mutational scanning revolutionizes the study of molecular recognition and protein-peptide interactions.
• High-throughput methods with non-canonical amino acids offer detailed insights into peptide behavior.
• These advancements are critical for optimizing de novo cyclic peptides for therapeutic applications.