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Published January 3, 2003 | Updated
Journal Article Open

De novo backbone and sequence design of an idealized α/β-barrel protein: evidence of stable tertiary structure


We have designed, synthesized, and characterized a 216 amino acid residue sequence encoding a putative idealized α/β-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 β-sheet surrounded by eight parallel α-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 222 nm and 208 nm revealed the presence of α-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 (ΔGH_2O) of 35 kJ/mol. Thermal unfolding monitored by near-UV CD revealed a cooperative transition with an apparent T_m of 65 °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 a/ b-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 α/β-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.

Additional Information

© 2002 Elsevier Science Ltd. Received 16 May 2002; received in revised form 18 October 2002; accepted 25 October 2002. Available online 3 December 2002. Edited by J. Thornton. We are grateful to D. Schaak and P. Osterhout of the Rowland Institute, B. Gordon and A. Street of Caltech for their very stimulating scientific interest and to A. Matagne, M. Galleni and M. Muller for critical reading of the manuscript. We thank A. Stern of the Rowland Institute for the backbone construction and N. Otthiers for peptide sequencing. F.O. is the recipient of a doctoral fellowship from the Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture (FRIA). This work was supported, in part, by European Space Agency grant number 12987/98/NL/VJ(IC).

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Updated - De_novo_Backbone_and_Sequence_Design_of_an_Idealized_alpha-beta-barrel_Protein-Evidence_of_Stable_Tertiary_Structure__Offredi_2003_.pdf



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