Radley, E. | bioRxiv Science

bioRxiv2026

A de novo CO2 Reductase Featuring a Cysteine-Ligated Cobalt Porphyrin Cofactor

Modern protein design methods based on deep learning allow generation of customized protein scaffolds with diverse geometries and functionalities. Here, we capitalize on these recent advances to develop hyper-thermostable de novo CO2 reductases featuring a cobalt porphyrin IX cofactor (CoPPIX). CoPPIX containing enzymes were assembled in vivo through media supplementation with cobalt salts and assessed for photocatalytic CO2 reductase activity. We identified two cysteine-ligated designs that exhibit high activity (>1000 turnovers at rates of up to 25 min-1) while suppressing competing hydrogen evolution pathways. A 2.1 [A] crystal structure shows close agreement to the design model with the Co-Cys bond programmed as intended. This study showcases the power of computational protein design in developing artificial enzymes to activate challenging molecules such as CO2.

biochemistry↗

bioRxiv2025

Long-read proteogenomic atlas of human neuronal differentiation reveals isoform diversity informing neurodevelopmental risk mechanisms

RNA splicing shapes neuronal identity and disease risk, yet current maps lack the developmental resolution and depth to resolve this complexity. Here, we integrate deep long-read RNA sequencing and proteomics in iPSC-derived cortical neurons to generate a high-resolution proteogenomic atlas of human neuron development. We identify 182,371 mRNA isoforms (over half previously unknown) and provide direct peptide evidence for the translation of hundreds of novel protein-coding sequences. Population genetics demonstrates that variants affecting novel exons and splice sites are under negative selection, underscoring the potential significance of these isoforms. During neuronal maturation, we observe that ASD risk genes undergo dynamic isoform switching, including microexon inclusion and intron retention, that remodel key protein domains and regulatory regions. Furthermore, we uncover widespread, long-range coordination between splicing and polyadenylation. Finally, our atlas enables variant reinterpretation in ASD, highlighting the value of an isoform-centric view for interpreting pathogenic variation in neurodevelopment.

bioinformatics↗