An expandable, modular de novo protein platform for precision redox engineering

The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme maquette protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.

Automated summary

The electron-conducting circuitry of life is a valuable, yet untapped resource in nanoscale biomolecular engineering. Researchers have characterized and analyzed a new diheme maquette protein, 4D2, and used it to create a modular platform for heme protein design. They redesigned a monoheme variant and validated its properties through experimental electrostatic redox potential calculations. 4D2 was then extended into a tetraheme helical bundle, demonstrating its potential as a molecular wire. This platform presents opportunities for redox protein design and the development of artificial electron-conducting circuitry in the future.