Octarellin VI: using rosetta to design a putative artificial (β/α)8 protein

Area of Science:

• Protein Engineering
• Computational Biology
• Structural Biology

Background:

• The Rosetta computational protein design protocol has demonstrated success in various protein engineering tasks.
• Designing de novo proteins with complex folds like the TIM-barrel presents a significant challenge due to the required number of residues.

Purpose of the Study:

• To evaluate the capability of the Rosetta protocol to design a de novo protein adopting the TIM-barrel fold.
• To engineer a novel protein, Octarellin VI, with approximately 216 residues, aiming for the TIM-barrel structure.

Main Methods:

• Application of the Rosetta computational protein design protocol.
• De novo design of a 216-residue protein (Octarellin VI).
• Characterization using far-UV and near-UV circular dichroism, tryptophan fluorescence, and dynamic light scattering.

Main Results:

• Octarellin VI was successfully designed and produced, exhibiting an amino acid composition similar to natural TIM-barrel proteins.
• Far-UV circular dichroism confirmed the presence of α-helical and β-sheet secondary structures, indicative of a folded protein.
• Tryptophan fluorescence and near-UV circular dichroism verified a stable tertiary structure, and the protein demonstrated heat stability up to 70°C.
• Dynamic light scattering indicated a particle size consistent with the designed model.

Conclusions:

• The study successfully created an artificial α/β protein exceeding 200 amino acids using computational design.
• Octarellin VI represents a significant advancement in de novo protein design, demonstrating the feasibility of engineering complex protein folds.
• Further optimization may be needed to address observed noncooperative chemical unfolding and low solubility.