De novo backbone and sequence design of an idealized alpha/beta-barrel protein: evidence of stable tertiary structure.
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.
Offredi
F
F
Laboratoire de Biologie Moléculaire et Génie Génétique, Université de Liège, B6, Sart Tilman, Belgium.
Dubail
F
F
Kischel
P
P
Sarinski
K
K
Stern
A S
AS
Van de Weerdt
C
C
Hoch
J C
JC
Prosperi
C
C
François
J M
JM
Mayo
S L
SL
Martial
J A
JA
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000235:genetics; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D011506:Proteins