Exploring Non-obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide-Protein...

Area of Science:

• Biochemistry
• Molecular Biology
• Medicinal Chemistry

Background:

• Glycogen phosphorylase a (GPa) is a key enzyme in glycogen metabolism.
• Targeting GPa is relevant for metabolic disease research.
• Polypeptide conjugates offer potential for modulating protein function.

Purpose of the Study:

• To investigate the effect of l-2-aminooctanoic acid (Aoc) incorporation on polypeptide conjugate affinity for GPa.
• To explore the potential of Aoc for peptide downsizing while maintaining binding affinity.
• To identify hydrophobic binding sites on protein surfaces using polypeptide conjugates.

Main Methods:

• Synthesis of 42-residue polypeptide conjugates with a small-molecule organic ligand targeting phosphorylated Ser15.
• Systematic replacement of hydrophobic amino acids (Nle, Ile, Leu) with Aoc.
• Affinity measurements using dissociation constant (K_D) values.
• Comparison of affinities between modified and unmodified polypeptide conjugates.
• Evaluation of "downsized" undecamer conjugates.

Main Results:

• Incorporation of Aoc residues increased binding affinity to GPa, reducing K_D from 280 nm to 27 nm for the 42-mer.
• Replacing specific hydrophobic residues with Aoc was crucial for this affinity enhancement.
• "Downsizing" the polypeptide to an undecamer with Aoc retained comparable affinity to the parent 42-mer.
• Hydrophobic binding sites on protein surfaces were identified by comparing Aoc-modified and unmodified conjugates.

Conclusions:

• Aoc residues provide a facile method for increasing affinity in protein recognition.
• Aoc-containing peptides can be "downsized" with minimal loss of binding affinity.
• Polypeptide conjugates are valuable tools for optimizing peptides and small molecules in biotechnology and biomedicine.