Characteristics of a de novo designed protein.
A series of 204 amino acid proteins intended to form TIM (triose phosphate isomerase) barrel structures were designed de novo. Each protein was synthesized by expression of the synthetic gene as a fusion protein with a portion of human growth hormone in an Escherichia coli host. After BrCN treatment, the protein was purified to homogeneity. The refolded proteins are globular and exist as monomers. One of the designed proteins is stable toward guanidine hydrochloride (GuHCl) denaturation, with a midpoint of 2.6 M determined from CD and tryptophan fluorescence measurements. The GuHCl denaturation is well described by a 2-state model. The NMR spectra, the thermal denaturation curves, and the 1-anilino-8-naphthalene sulfonic acid binding imply that the stability of the protein arises mainly from hydrophobic interactions, which are probably of a nonspecific nature. The protein has a similar shape to that of rabbit triosephosphate isomerase, as determined by electron microscopy.
Tanaka
T
T
Protein Engineering Research Institute, Osaka, Japan.
Kimura
H
H
Hayashi
M
M
Fujiyoshi
Y
Y
Fukuhara
K
K
Nakamura
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004274:DNA, Recombinant / Q000235:genetics; D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D013006:Growth Hormone / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics
D004274:DNA, Recombinant; D011506:Proteins; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; D014305:Triose-Phosphate Isomerase