Silberg, Joff2020-09-222020-09-222020-122020-09-22December 2Truong, Albert Tran. "Engineering protein dependence into ferredoxin-based electron transfer systems." (2020) Master’s Thesis, Rice University. <a href="https://hdl.handle.net/1911/109362">https://hdl.handle.net/1911/109362</a>.https://hdl.handle.net/1911/109362Electron flow is central to bioenergetics and life, but our ability to control this biological process remains limited. One method of controlling electron flow involves engineering protein electron carriers (PECs) into switches with electron transfer activity that can be actuated under specific conditions. Previous work has created ferredoxins (low-potential PECs) whose activity depends on the presence of specific small molecules by splitting a ferredoxin and fusing the resulting fragments to a pair of proteins that dimerize in the presence of a small molecule or to the termini of a ligand binding domain. These approaches are limited to the specific molecules that can bind these domains to toggle ferredoxin activity. To expand the molecular repertoire for controlling electron transfer, I propose two strategies for constructing ferredoxins whose activity depends on an arbitrary molecule. The first strategy involves fusing a pair of nanobodies which share the same binding target to a pair of split ferredoxin fragments. The presence of the binding target is expected to cause both nanobodies to come together, allowing the ferredoxin fragments to complement and resume ferredoxin activity. To explore this strategy, three different anti-GFP nanobodies were fused in a variety of configurations to a ferredoxin fragment, and GFP was fused to the complementary ferredoxin fragment. Ferredoxin activity was observed when specific configurations of nanobody-ferredoxin and GFP-ferredoxin fusions were co-expressed, indicating the binding of an anti-GFP nanobody to its target can restore ferredoxin activity. However, when two different anti-GFP nanobodies were fused to a pair of split ferredoxin fragments, the presence of free GFP was unable to toggle ferredoxin activity. The second strategy involves inserting a marginally stable nanobody domain into a ferredoxin, which is unfolded and disrupts ferredoxin activity in the absence of its target protein, but is folded and restores ferredoxin activity in the presence of its target protein. To explore this strategy, three different stable anti-GFP nanobodies were inserted into a ferredoxin, and two of the three were found not to disrupt ferredoxin activity, indicating that these variants are potential targets for laboratory evolution for the desired switch behavior. Successful engineering of chemical-dependent ferredoxin switches may lead to the creation of cellular sensors with electrical output that can interface with electronic devices.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.ferredoxinnanobodyelectron transferThesis2020-09-22