title: A Fluorescent Readout for the Oxidation State of Electron Transporting Proteins in Cell Free Settings.
abstract: Pathways involving sequential electron transfer between multiple proteins are ubiquitous in nature. Here, we demonstrate a new class of fluorescent protein-based reporters for monitoring electron transport through such multistage cascades, specifically those involving ferredoxin-like electron transporters. We created protein fusions between mammalian Adrenodoxin (Adx) and plant Ferredoxin (Fdx) with fluorescent proteins of different colors and found that the fluorescence of such fusions is highly sensitive to the redox state of the electron transporter. The increase in fluorescence from the oxidized to the reduced state was inversely proportional to the linker length between the fusion partners. We first used our approach to quantitatively characterize electron transfer from NADPH through Adrenodoxin Reductase (AdR) to Adrenodoxin (Adx). Our data allowed us to build a detailed mathematical model of this mitochondrial electron transfer chain and validate previously proposed mechanisms. Then, we showed that an Adx-GFP fusion could serve as a sensor for the activity of bacterial Type I Cytochrome P450s (CYPs), a very large class of enzymes with important roles in biotechnology. We further showed that fluorescence of a direct fusion between CYP and GFP was sensitive to CYP activity, suggesting that our approach is applicable to an even broader class of proteins, which undergo a redox state change during their work cycle.
authors.affiliation: Department of Electrical Engineering, University of Washington , Seattle, 98195 Washington, United States.
authors.forename: Sergii
authors.initials: S
authors.lastname: Pochekailov
authors.affiliation: Department of Electrical Engineering, University of Washington , Seattle, 98195 Washington, United States.
authors.forename: Rebecca R
authors.initials: RR
authors.lastname: Black
authors.affiliation: Department of Electrical Engineering, University of Washington , Seattle, 98195 Washington, United States.
authors.forename: Venkata Pramod
authors.initials: VP
authors.lastname: Chavali
authors.affiliation: Department of Electrical Engineering, University of Washington , Seattle, 98195 Washington, United States.
authors.forename: Arjun
authors.initials: A
authors.lastname: Khakhar
authors.affiliation: Department of Electrical Engineering, University of Washington , Seattle, 98195 Washington, United States.|Department of Computer Science and Engineering, University of Washington , Seattle, 98195 Washington, United States.
authors.forename: Georg
authors.initials: G
authors.lastname: Seelig
mesh_terms: D000325:Adrenodoxin / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005288:Ferredoxins / Q000235:genetics* / Q000378:metabolism; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008962:Models, Theoretical; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D000072516:Retinoic Acid 4-Hydroxylase / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005288:Ferredoxins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D000325:Adrenodoxin; D049452:Green Fluorescent Proteins; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; C517094:15beta-hydroxylase CYP106A2, Bacillus megaterium; D000072516:Retinoic Acid 4-Hydroxylase; D005287:Ferredoxin-NADP Reductase
keywords: Cytochrome P450; GFP sensor; enzyme activity sensing; fluorescent protein