Second-generation octarellins: two new de novo (β/α)₈ polypeptides designed for investigating the influence of β-residue packing on the α/β-barrel structure stability

Abstract

The sequence of octarellin I, the first de novo (beta/alpha)8 polypeptide, was revised according to several criteria, among others the symmetry of the sequence, beta-residue volume and hydrophobicity, and charge distribution. These considerations and the overall conclusions drawn from the first design led to two new sequences, corresponding to octarellins II and III. Octarellin II retains perfect 8-fold symmetry. Octarellin III has the same sequence as octarellin II, except for the beta-strands which exhibit a 4-fold symmetry. The two proteins were produced in Escherichia coli. Infrared and CD spectral analyses of octarellins II and III reveal a high secondary structure content. Non-denaturing gel electrophoresis, molecular sieve chromatography and analytical ultracentrifugation suggest that both of these second-generation artificial polypeptides exist as a mixture of a monomer and a dimer form. Octarellins II and III are at least 10 times more soluble than octarellin I. Urea-induced unfolding followed by fluorescence emission suggests that the tryptophan residues, designed to be buried in the (beta/alpha)8, are indeed packed in the hydrophobic core of both proteins. However, octarellin III displays a higher stability towards urea denaturation, indicating that introducing 4-fold symmetry into the beta-barrel might be important for stability of the overall folding.

Insights into the Fold Organization of TIM Barrel from Interaction Energy Based Structure Networks

“…All these results suggest that (a) the β barrel formation driven by high–energy interactions (with the participating residues being conserved) seem to be an important step in the organization of the TIM barrel; (b) the formation of the other interfaces mainly by low–energy interactions (with residue conservation being immaterial) is a more canonical step in the fold formation common to all the folds of the α/β class, and can be taken up by a variety of sequences, thus contributing the high sequence diversity. These conclusions concur with several experimental observations that suggest that while the α/β interfaces in TIM are resilient to mutations the β barrel is sensitive [18], [40], [41], [44].…”

Reverse engineering the (β/α) 8 barrel fold

“…Taken further, one might infer that novel barrel structures could be engineered by appropriately patterning hydrophobic and polar residues in an amino acid sequence. Five attempts to design (␤͞␣) 8 barrel proteins by H͞P patterning have been reported (8,9). All of the designs produced barrels with ill-defined and fluctuating tertiary structures.…”

The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools

“…Work in our lab, based on various approaches, has yielded several generations of Octarellins (Beauregard et al, 1991;Figueroa et al, 2013;Goraj et al, 1990;Houbrechts et al, 1995), but solubility and structural stability issues have prevented us from determining the exact structure of any of them. Although the secondary structure of one of the previous version, Octarellin V, described in 2003 (Offredi et al, 2003), seemed compatible with the in silico model, this protein failed to meet the technical requirements for NMR spectroscopy and X-ray diffraction.…”