De novo Backbone and Sequence Design of an Idealized α/β-barrel Protein: Evidence of Stable Tertiary Structure

Abstract

We have designed, synthesized, and characterized a 216 amino acid residue sequence encoding a putative idealized alpha/beta-barrel protein. The design was elaborated in two steps. First, the idealized backbone was defined with geometric parameters representing our target fold: a central eight parallel-stranded beta-sheet surrounded by eight parallel alpha-helices, connected together with short structural turns on both sides of the barrel. An automated sequence selection algorithm, based on the dead-end elimination theorem, was used to find the optimal amino acid sequence fitting the target structure. A synthetic gene coding for the designed sequence was constructed and the recombinant artificial protein was expressed in bacteria, purified and characterized. Far-UV CD spectra with prominent bands at 222nm and 208nm revealed the presence of alpha-helix secondary structures (50%) in fairly good agreement with the model. A pronounced absorption band in the near-UV CD region, arising from immobilized aromatic side-chains, showed that the artificial protein is folded in solution. Chemical unfolding monitored by tryptophan fluorescence revealed a conformational stability (DeltaG(H2O)) of 35kJ/mol. Thermal unfolding monitored by near-UV CD revealed a cooperative transition with an apparent T(m) of 65 degrees C. Moreover, the artificial protein did not exhibit any affinity for the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS), providing additional evidence that the artificial barrel is not in the molten globule state, contrary to previously designed artificial alpha/beta-barrels. Finally, 1H NMR spectra of the folded and unfolded proteins provided evidence for specific interactions in the folded protein. Taken together, the results indicate that the de novo designed alpha/beta-barrel protein adopts a stable three-dimensional structure in solution. These encouraging results show that de novo design of an idealized protein structure of more than 200 amino acid residues is now possible, from construction of a particular backbone conformation to determination of an amino acid sequence with an automated sequence selection algorithm.

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.…”

“…More than ten years ago, in our attempt to address the inverse folding problem, we designed the artificial protein Octarellin V (Offredi et al, 2003). This protein displayed promising features as it was not a molten globule and its secondary structure content was compatible with the in silico design.…”

“…Octarellin V was purified from inclusion bodies as it was previously reported (Offredi et al, 2003). Octarellin V.1 was produced in BL21(DE3) at 37°C after 4 hours of induction (IPTG 1 mM).…”

Designing New Proteins

“…The simplest way to find a backbone structure that is capable of being designed is to reuse experimentally solved backbone structures and/or local loop remodeling; however, there has been work on methods to design de novo backbone structures [17][18][19][20].…”

Optimal Attachment Position and Linker Length Promote Native‐like Character of Cavitand‐Based Template‐Assembled Synthetic Proteins (TASPs)

“…The near-UV region of the CD spectra, however, can yield information about the tertiary structure of the protein if a chromophore is present-again, in this case the cavitand template. [21] An aromatic absorption in the near-UV region can be observed in the presence of non-averaged structural elements. Molten-globule structures typically show an absence or reduction of signals in the near-UV region because of their time-averaged fluctuations.…”