Abstract
The computational protein design protocol Rosetta has been applied successfully to a wide variety of protein engineering problems. Here the aim was to test its ability to design de novo a protein adopting the TIM-barrel fold, whose formation requires about twice as many residues as in the largest proteins successfully designed de novo to date. The designed protein, Octarellin VI, contains 216 residues. Its amino acid composition is similar to that of natural TIM-barrel proteins. When produced and purified, it showed a far-UV circular dichroism spectrum characteristic of folded proteins, with α-helical and β-sheet secondary structure. Its stable tertiary structure was confirmed by both tryptophan fluorescence and circular dichroism in the near UV. It proved heat stable up to 70°C. Dynamic light scattering experiments revealed a unique population of particles averaging 4 nm in diameter, in good agreement with our model. Although these data suggest the successful creation of an artificial α/β protein of more than 200 amino acids, Octarellin VI shows an apparent noncooperative chemical unfolding and low solubility.
Highlights
The Inverse Protein-folding Problem The aim of de novo protein design, often called the ‘‘inverse protein-folding problem’’, is to find amino acid sequences compatible with a given protein tertiary structure
For construction of loop regions, six-residue fragments of Protein Data Bank (PDB) proteins displaying the secondary structure pattern [E,E/ L,L,L,L,L,L/H,H] for ba-loops or [H,H/L,L,L,L,L,L/E,E] for ab-loops were extracted with the Rosetta loop-building protocol [36]
Models taken from one cycle to the were selected by application of a filter
Summary
The Inverse Protein-folding Problem The aim of de novo protein design, often called the ‘‘inverse protein-folding problem’’, is to find amino acid sequences compatible with a given protein tertiary structure. Solving the inverse protein-folding problem is a stringent test of our understanding of sequence-structure relationships in proteins. Improving this understanding should help to solve the ‘‘protein-folding problem’’ per se: predicting what tertiary structure a given amino acid sequence will adopt. This should enable us to engineer proteins with custom functions and properties. Reported successes in designing large artificial proteins involved creating new proteins by assembling, in variable number, multiple copies of a same motif of no more than 40 amino acids long. [3,4]
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