De Novo Design, Synthesis, and Function of Semiartificial Myoglobin Conjugated with Coiled‐Coil Two‐α‐Helix Peptides

Abstract

The introduction of a flavin chromophore on the myoglobin (Mb) surface and an effective electron-transfer (ET) reaction through the flavin were successfully achieved by utilizing the self-assembly of heterostranded coiled-coil peptides. We have prepared a semiartificial Mb, named Mb-1alphaK, in which an amphiphilic and cationic alpha-helix peptide is conjugated at the heme propionate (Heme-1alphaK). Heme-1alphaK has a covalently bound iron-protoporphyrin IX (heme) at the N terminus of a 1alphaK peptide sequence. This sequence was designed to form a heterostranded coiled-coil in the presence of a counterpart amphiphilic and anionic 1alphaE peptide sequence in a parallel orientation. Two peptides, Fla(1)-1alphaE and Fla(31)-1alphaE, both incorporating a 10-methylisoalloxazine moiety as an artificial flavin molecule, were also prepared (Fla=2-[7-(10-methyl)isoalloxazinyl]-2-oxoethyl). Heme-1alphaK was successfully inserted into apomyoglobin to give Mb-1alphaK. Mb-1alphaK recognized the flavin-modified peptides and a two-alpha-helix structure was formed. In addition, an efficient ET from reduced nicotinamide adenine dinucleotide to the heme center through the flavin unit was observed. The ET rate was faster in the presence of Fla(1)-1alphaE than in the presence of Fla(31)-1alphaE or the equivalent molecule that has no peptide chain. These results demonstrate that the introduction of a functional chromophore on the Mb surface can be achieved by using specific peptide-peptide interactions. Moreover, the dependence of the ET rate on the position of the flavin indicated that the distance between the heme active site and the flavin chromophore was regulated by the three-dimensional structure of the designed polypeptide.

Coiled coils: attractive protein folding motifs for the fabrication of self-assembled, responsive and bioactive materials

“…Sakamoto et al used a highly specific, parallel heterodimer, 1aE/1aK, as a non-covalent linker to conjugate a chromophore (flavin) to the myoglobin surface. 148 The conjugation takes place via the self-assembly of modified coiled coil forming peptides, a heme-1aK modified peptide and a flavin-1aK, followed by association of myoglobin with the heme to form a non-covalent complex. Flavin chromophore was used to mimic a natural cofactor.…”

14 Bioinspired organic chemistry

“…The successful design of peptides capable of mimicking protein-protein interactions has advanced to the productive generation of novel protein interaction sites based on artificial scaffolds, 29,30 and of new proteins capable of new functions. [31][32][33] A more functional approach involves the mutagenesis of scaffolds with convergent loop regions, 34 reminiscent of antibody optimisation. A variation in the formation of proteins capable of recognition at the surface of another protein is to use an active site based interaction to guide the initial interaction.…”