Inside-Out Design of Zinc-Binding Proteins with Non-Native Backbones.

Area of Science:

• Protein Engineering
• Computational Biology
• Biochemistry

Background:

• Designing functional proteins requires precise control over tertiary structure and the creation of molecular binding sites.
• Existing methods for *de novo* protein design face challenges in achieving specific structural and functional outcomes.

Purpose of the Study:

• To develop and validate a new computational strategy for the *de novo* design of functional proteins with specified binding sites.
• To engineer novel zinc-binding proteins using an inside-out design approach.

Main Methods:

• Utilized the Rosetta molecular modeling program with an 'inside-out' design strategy, starting with ligand-contacting α-helices.
• Built full-sized proteins around a ligand by adding helices to form a stable core and enhance ligand interactions.
• Designed 12 zinc-binding proteins with 4-5 helices each.

Main Results:

• Four of the 12 designed proteins successfully folded and exhibited zinc-binding capabilities.
• Observed equilibrium dissociation constants (K_d) for zinc binding ranged from 95 nM to 1.1 μM.
• The highest-affinity design (N12) adopted a unique conformation, validated by NMR, and closely matched its AlphaFold model (RMSD < 1 Å).

Conclusions:

• The inside-out design strategy in Rosetta is effective for creating functional, folded proteins with specific ligand-binding properties.
• Computational predictions, including AlphaFold, can guide and validate *de novo* protein designs, although sequence-level packing remains a challenge.
• This approach advances the field of protein engineering for creating novel biomolecules with tailored functions.