Engineering Naturally Occurring Multi-Heme Cytochrome Nanowires into Self-Assembled Nanogels

The development of advanced biomaterials capable of electrical signal transmission is vital for regenerative medicine and bioelectronics, with injectable conductive nanogels showing significant promise. While naturally occurring, biocompatible extracellular conductive nanowires (ECNs) from anaerobic bacteria, such as the multi- heme cytochrome proteins, offer a compelling solution to the limitations of synthetic materials (e.g., solubility and biocompatibility), their widespread application is currently limited by challenges in their rational engineering and efficient production. Specifically, the recently discovered ECN protein family has not yet been integrated into recent novel AI protein design tools, and their complex in vivo assembly mechanisms remain poorly understood. This proposal will bridge these gaps by first identifying and engineering OmcE cytochrome nanowires that form large, ordered bundles. This involves comprehensive large-scale genomic and AlphaFold3-guided virtual screens to pinpoint novel OmcE homologs, followed by high-resolution cryo-EM characterization to elucidate their structural details and bulk conductivity measurements to confirm electrical properties. Subsequently, state- of-the-art AI tools will be employed to engineer novel OmcE variants exhibiting robust self-assembly into advanced conductive nanogels. Simultaneously, another major objective is to visualize the OmcE secretion system in situ to unravel its intricate assembly mechanism. We hypothesize that these nanowires assemble via a large outer membrane porin, analogous to the chaperone-usher pathway. Sub-tomogram averaging will be utilized to reconstruct the porin's structure at sub-nanometer resolution, providing critical molecular blueprints for the rational design of OmcE variants with enhanced self-assembly properties and enabling their efficient recombinant overexpression. Ultimately, this work will facilitate the precise engineering of "super" OmcE nanowires for conductive nanogels, offering transformative insights into their biosynthesis and establishing a foundation for a new generation of protein-based bioelectronic materials. Project Number: 1F31NS149527-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Holly Petersen | Institution: UNIVERSITY OF ALABAMA AT BIRMINGHAM, BIRMINGHAM, AL | Award Amount: $43,714 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F04B-S (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11382503