Analyzing the effects of random mutations on ferredoxin-mediated electron transfer using a cellular assay
| dc.contributor.advisor | Silberg, Jonathan J | en_US |
| dc.creator | Bluford, Jordan Terrell | en_US |
| dc.date.accessioned | 2023-08-09T19:19:46Z | en_US |
| dc.date.available | 2023-08-09T19:19:46Z | en_US |
| dc.date.created | 2023-05 | en_US |
| dc.date.issued | 2023-04-21 | en_US |
| dc.date.submitted | May 2023 | en_US |
| dc.date.updated | 2023-08-09T19:19:46Z | en_US |
| dc.description.abstract | Ferredoxins are iron-sulfur cluster containing proteins, which are thought to be one of the oldest families of metalloproteins. These protein electron carriers support a wide range of metabolic processes, ranging from photosynthesis and amino acid synthesis to assimilation of nitrogen and carbon. They are also ubiquitous across the domains of life with several different paralogs and metallocluster geometries being found in archaea, bacteria, and eukaryotes. Despite the abundance of biochemical and biophysical information available on these proteins, our understanding of their structure-redox relationships is not yet sufficient to anticipate their control over electron flow in cells. In part, this knowledge gap exists because we cannot score the contribution that each ferredoxin residue makes to in vivo functions. To better understand ferredoxin electron transfer in cells, I used laboratory evolution to study sequence-structure-redox relationships in Zea mays ferredoxin-3, a 2Fe-2S protein. To select for mutant electron transfer, I expressed this ferredoxin in a synthetic, linear-electron transfer pathway and showed that this protein complemented the growth defect when fused to a red fluorescent protein (mRFP). I then computationally designed a site-saturation mutagenesis library, which was designed to encode all single amino acid substitutions of ferredoxin-3. This library was characterized, selected for ferredoxin-mediated electron transfer, and the fitness of each mutant variant was scored. This study establishes the effects of direct fusion of fluorescent proteins on ferredoxin electron transfer and how the use of a flexible glycine-serine linker affects electron transfer. It also illustrates how a mutant library can be selected to gain information about the sequence-structure-redox relationship within 2Fe-2S ferredoxins and to probe the contribution of each amino acid residue to ferredoxin-partner interactions in a synthetic pathway. This approach can be extended to diverse ferredoxins, homologs and partner proteins. A better understanding of redox-insulated ferredoxin pathways will support future efforts to control the electron fluxome for metabolic engineering, electrosynthesis, and protein-based biosensors. | en_US |
| dc.format.mimetype | application/pdf | en_US |
| dc.identifier.citation | Bluford, Jordan Terrell. "Analyzing the effects of random mutations on ferredoxin-mediated electron transfer using a cellular assay." (2023) Diss., Rice University. <a href="https://hdl.handle.net/1911/115184">https://hdl.handle.net/1911/115184</a>. | en_US |
| dc.identifier.uri | https://hdl.handle.net/1911/115184 | en_US |
| dc.language.iso | eng | en_US |
| dc.rights | Copyright 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. | en_US |
| dc.subject | ferredoxin | en_US |
| dc.subject | site-saturation mutagenesis | en_US |
| dc.title | Analyzing the effects of random mutations on ferredoxin-mediated electron transfer using a cellular assay | en_US |
| dc.type | Thesis | en_US |
| dc.type.material | Text | en_US |
| thesis.degree.department | Biochemistry and Cell Biology | en_US |
| thesis.degree.discipline | Natural Sciences | en_US |
| thesis.degree.grantor | Rice University | en_US |
| thesis.degree.level | Doctoral | en_US |
| thesis.degree.name | Doctor of Philosophy | en_US |
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