Hyperstable Synthetic Mini-Proteins as Effective Ligand Scaffolds

Area of Science:

• Protein engineering
• Biophysical chemistry
• Molecular biology

Background:

• Small, single-domain protein scaffolds offer advantages for molecular binding ligands, including efficient transport and modularity.
• Mini-proteins require a balance between stability and diversity for novel functions.

Purpose of the Study:

• To assess the developability and evolvability of synthetic 40-amino acid βαββ protein variants.
• To identify robust protein scaffolds for generating high-affinity binders.

Main Methods:

• Designed and synthesized 43 libraries of βαββ protein variants with diversified paratopes (sheet, loop, helix).
• Screened millions of variants for binding to seven different targets.
• Assessed protein stability (Tm) and soluble expression.

Main Results:

• Discovered a scaffold library yielding hundreds of binders to seven targets with 6-122 nM affinities.
• Achieved average melting temperatures (Tm) of ≥78 °C, indicating high stability.
• Identified sheet paratopes as most consistently developable and framework 1 as most evolvable.

Conclusions:

• Certain βαββ protein scaffolds are highly developable and evolvable, serving as excellent starting points for engineering potent binders.
• The discovered binders exhibit high affinity and stability without requiring affinity maturation.
• Paratope evolvability is target-dependent, but some libraries show broad applicability.