Synthesis, purification and initial structural characterization of octarellin, a de novo polypeptide modelled on the α/β-barrel Proteins

Abstract

We have attempted to construct an artificial polypeptide that folds like the eight-stranded parallel beta-barrel structures. Our approach consists of repeating eight times a unit peptide designed to adopt a 'beta-strand/alpha-helix' pattern. A first 'test' sequence for this structural unit was deduced from a series of parameters defined after an analysis of three natural alpha/beta-barrel proteins and including principally the lengths of the secondary structure elements, the alpha/beta packing and the fitting on average Garnier profiles. The gene encoding this structural unit was synthesized, cloned and expressed in Escherichia coli either as a monomer or as direct repeats of 2-12 units. Preliminary structural characterization of the 7-, 8- and 9-fold unit polypeptides by circular dichroism measurements indicates the presence of the predicted amount of alpha-helix in the three proteins. Further analysis by urea-gradient gel electrophoresis demonstrates that, in the conditions tested, only the 8-fold unit polypeptide forms a compact structure through a cooperative and rapid two-state folding transition involving long-range molecular interactions.

Changing the Structural Context of a Functional β-Hairpin

“…1), superimposable with little differences in the spatial disposition of their backbone atoms. Second, although Hynes et al (68) and Wolfson et al (12) showed that loops can be transferred from one structural context to another as structural 4 E. Drakopoulou and C. Vita, unpublished result.…”

“…After the third booster immunization, on day 100, the serum titer (in both immunized rabbits) was quite high and close to 10 6 . This high immunogenic response may look surprising from such a small molecule, but its high conformational stability, which may increase its t1 ⁄2 and maintain a high concentration in the blood, and the possibility that the scorpion scaffold may contain epitope inducing T cell help, as shown by the even higher immunogenicity of native charybdotoxin, 4 may explain this experimental finding.…”

“…Simple protein architectures or scaffolds (1)(2)(3)(4)(5)(6) have been obtained by de novo protein design, but only a few have been harnessed with a function, such as in the case of the heme-binding and redox-active proteins (7,8) and of the DNA-binding proteins (9,10). Genuine successes in the generation of functionally useful novel proteins, however, have been obtained by a more conservative approach which exploits the structure of some natural proteins as appropriate structural scaffolds and manipulates surface loops and insert new functional sequences in permissive regions.…”

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.…”

A quantitative methodology for the de novo design of proteins

“…Several de novo protein design experiments have already sought alternative amino acid sequences for well-studied folds (Sander et al, 1992b; for a review, see DeGrado & Matthews, 1993), such as the 4-helix bundle (Regan & DeGrado, 1988;Hecht et al, 1990;Hill et al, 1990;Sander et al, 1992a;Schafmeister et al, 1993), @-bell structures (Richardson & Richardson, 1987), and a/@-barrels (Hubbard & Blundell, 1989;Goraj et al, 1990). In a similar manner, folds not found naturally, such as an "open sandwich," have been designed (Fedorov et al, 1992).…”