Reference : De novo backbone and sequence design of an idealized alpha/beta-barrel protein: Evide...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/12069
De novo backbone and sequence design of an idealized alpha/beta-barrel protein: Evidence of stable tertiary structure
English
Offredi, Fabrice [Université de Liège - ULG > Département des Sciences Biomédicales et Précliniques > Biologie Moléculaire et Génie Génétique > >]
Dubail, Fabien [Université de Liège - ULG > Département des sciences biomédicales et précliniques > Biologie Moléculaire et Génie Génétique > >]
Kischel, Philippe mailto [Université de Liège - ULg > Département des sciences biomédicales et précliniques > Biologie Moléculaire et Génie Génétique > >]
Sarinski, K. [> > > >]
Stern, A. S. [> > > >]
Van de Weerdt, Cécile [Université de Liège - ULg > Département des sciences de la vie > Biologie Moléculaire et Génie Génétique > >]
Hoch, J. C. [> > > >]
Prosperi, Christelle [Université de Liège - ULg > Département de physique > Département de physique >]
François, Jean-Marie [Université de Liège - ULg > Département des sciences biomédicales et précliniques > Biologie Moléculaire et Génie Génétique > >]
Mayo, S. L. [> > > >]
Martial, Joseph [Université de Liège - ULg > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire >]
3-Jan-2003
Journal of Molecular Biology
Academic Press Ltd Elsevier Science Ltd
325
1
163-174
Yes (verified by ORBi)
International
0022-2836
London
[en] protein design ; backbone parameterization ; side-chain modeling ; fluorescence ; circular dichroism
[en] 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.
Researchers ; Professionals ; Students
http://hdl.handle.net/2268/12069
10.1016/S0022-2836(02)01206-8
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WK7-47C3H5G-N&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=5a1d51464c9786c0fc34036405896284

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