Chemical Evolution of De novo Designed Artificial Proteins Incorporating Biological Functions

The chemical evolution of four-α-helix bundle polypeptide to a flavoenzyme model was attempted by designing single-chain53-peptides。The flavoenzyme models were examined for catalytic oxidation reaction in aqueous solution.Since the hydrophobic core seemed to be too tightly aggregated for atalytic group，we tested a series of alkane-sulfonates to enlarge it by forming mixed micelles。The expanded hydrophobic core may more easily accommodate the hydrophobic substrate.The alkyl chain length longer than dodecyl enhanced the oxidation of benzyl-NAH by a flavoenzyme model by about6-fold。These results suggest that the hydrophobic core in the bundle structure is useful to place the catalytic groups and have the advantage of accommodating hydrophobic substrates.On the other hand，a porphyrin derivative was incorporated into a single-chained two-α-helix polypeptide containing29 amino acid residues via the thiol side chain of Cys residue。The dimerized Fe(III)-porphyrin-linked two-α-helix…More polypeptide was examined for the biomimetic peroxidase-like activity with H I D 22 ii D 2 O I D 22 ii or 3-chloroperbenzoic acid(MCPBA)as the oxidant.The k I D2 cat自己D2/K I D 2 M I D 2 value for the oxidation by the polypeptide with mCPBA was increased by 5000 times than that with H I D 22 ii D 2.Thus，we demonstrated the possibility to utilize the hydrophobic core of the four-α-helix bundle structure to evolve the artificial proteins chemically by de novo design to the artificial enzymes.Then，we chose aβαβα-structure as a polypeptide scaffold，since theβα-unit is found in TIM barrel。In order to place a porphyrin ring in the hydrophobic space in the folded structure，we designed a single-chained49-peptide applying the amphiphilicα-helix andβ-strand motifs.Peroxidase-like oxidation activity of this polypeptide occurred specifically in the presence of LPO rather than H I D22 ii D 2 O I D 22 ii at low methanol content，reflecting that the hydrophobic inside was used as a binding site of the oxidant.We concluded that the de novo design of artificial proteins should be further polished to enhance their catalytic activities.Less：Less

The chemical evolution of four-α-helix bundle polypeptide to a flavoenzyme model was attempted by designing single-chain 53-peptides. The flavoenzyme models were examined for catalytic oxidation reaction in aqueous solution. Since the hydrophobic core seemed to be too tightly aggregated for atalytic group, we tested a series of alkane-sulfonates to enlarge it by forming mixed micelles. The expanded hydrophobic core may more easily accommodate the hydrophobic substrate. The alkyl chain length longer than dodecyl enhanced the oxidation of benzyl-NAH by a flavoenzyme model by about 6-fold. These results suggest that the hydrophobic core in the bundle structure is useful to place the catalytic groups and have the advantage of accommodating hydrophobic substrates. On the other hand, a porphyrin derivative was incorporated into a single-chained two-α-helix polypeptide containing 29 amino acid residues via the thiol side chain of Cys residue. The dimerized Fe(III)-porphyrin-linked two-α-helix … More polypeptide was examined for the biomimetic peroxidase-like activity with HィイD22ィエD2 OィイD22ィエD2 or 3-chloroperbenzoic acid (mCPBA) as the oxidant. The kィイD2catィエD2/KィイD2MィエD2 value for the oxidation by the polypeptide with mCPBA was increased by 5000 times than that with HィイD22ィエD2OィイD22ィエD2.Thus, we demonstrated the possibility to utilize the hydrophobic core of the four-α-helix bundle structure to evolve the artificial proteins chemically by de novo design to the artificial enzymes. Then, we chose a βαβα-structure as a polypeptide scaffold, since the βα-unit is found in TIM barrel. In order to place a porphyrin ring in the hydrophobic space in the folded structure, we designed a single-chained 49-peptide applying the amphiphilic α-helix and β-strand motifs. Peroxidase-like oxidation activity of this polypeptide occurred specifically in the presence of LPO rather than HィイD22ィエD2 OィイD22ィエD2 at low methanol content, reflecting that the hydrophobic inside was used as a binding site of the oxidant. We concluded that the de novo design of artificial proteins should be further polished to enhance their catalytic activities. Less