Browsing by Author "Silberg, Jonathan J"

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    Analyzing the effects of random mutations on ferredoxin-mediated electron transfer using a cellular assay
    (2023-04-21) Bluford, Jordan Terrell; Silberg, Jonathan J
    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.
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    Near-infrared fluorescent protein reporters for imaging iron-sulfur cluster binding to human glutaredoxin 2
    (2017-07-21) Lie, Margaret; Silberg, Jonathan J
    Evolutionarily, iron-sulfur (Fe-S) proteins have been vital for cellular functioning and the persistence of life. These Fe-S proteins typically consist of 2Fe-2S or 4Fe-4S clusters, which perform roles such as electron transfer and catalysis. Processes such as metabolism, respiration, and DNA synthesis rely on these proteins to drive their progress. The homeostasis of Fe-S proteins and their clusters impacts the growth and overall health of most organisms. Consequently, defects in these proteins result in detrimental diseases such as anemia, ataxia, and myopathy. Similar diseases arise when defects exist in the biogenesis machinery of the Fe-S clusters themselves. However, we currently cannot use imaging to assess the Fe-S cluster levels on these proteins within animals. My thesis research aims to develop new tools for imaging Fe-S proteins by using fragmented fluorescent proteins with excitation and emission in the near-infrared region of the spectrum. Using human glutaredoxin 2 (GRX2), I created a dozen biosensors by fusing fragments of a split near-infrared fluorescent protein (IFP) to this dimerizing dithiol glutaredoxin. My results indicate that Escherichia coli expressing IFP fragments fused to GRX2 display significantly higher fluorescence than cells expressing the same IFP fragments fused to a C37A mutant of GRX2, which lacks the active site cysteine necessary for coordination to a 2Fe2S cluster and dimerization. Endpoint assays were used to show the differences in biosensor behavior at two different temperatures. The biosensors behave differently at 37°C and 25°C. At reduced temperatures, the C37A mutant of GRX2 fluoresce to a greater extent, which may arise from dimerization without binding to a 2Fe2S cluster. In addition, I showed that dynamic measurements with these biosensors display differences in signal over time, and these assays could be easily used to analyze the behavior of GRX2 in different cysteine desulfurase knockout strains. The split IFP fused to GRX2 will be applicable when investigating 2Fe2S cluster availability in various microorganisms and multicellular organisms. The results of my thesis work have yielded a new imaging tool that can be used to advance the understanding of how genetics and environment alter the construction of 2Fe2S clusters and affect dimerization of Fe-S proteins. Finally, comparisons of biosensor expression will enhance the understanding of disease states due to defects in mutations in Fe-S proteins or the Fe-S biogenesis machinery.
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    Using gas-producing enzymes to enable bacterial reporting within environmental matrices
    (2018-03-30) Cheng, Hsiao-Ying; Silberg, Jonathan J
    Microbes drive processes in the Earth system far exceeding their physical scale, mediating significant fluxes in biogeochemical cycles. Microbial behavior also affects soil development, water quality, and crop yields. The tools of synthetic biology have the potential to significantly improve our understanding of the roles that microbes play in these processes and the effects of environmental fluctuations on microbial behaviors, which can advance our ability to engineer microbial system for environmental applications, such as bioremediation, waste water treatment, and engineered rhizosphere. However, synthetic biology has not yet been widely used within environmental materials (soils, sediments, and biomass). One of the challenges is that there is a lack of robust and simple-to-detect reporter proteins for nontransparent and heterogeneous materials. Common genetic reporters used to read out circuit status have limited utility for in situ measurements in Earth materials because environmental matrices display high absorbance and auto-fluorescence at wavelengths of light used for visual reporters like GFP. This technical limitation has made it challenging to use programmed microbes to study how variation in soil environmental parameters (moisture, nutrient status, mineralogy, structure, and temperature) affect real-time biological behaviors. To overcome this limitation, my thesis research aims to develop a new reporting strategy using gas-producing enzymes, which generate diffusible gases that can be quantified in the headspace of soils using gas chromatography. First, I characterized the activities of two gas-producing enzyme, methyl halide transferase (MHT) and ethylene forming enzyme (EFE), in liquid media and an agricultural soil. Using these two enzymes, gas reporting strains were developed to monitor two dynamic soil microbial processes in situ, horizontal gene transfer and quorum sensing. These proof-of-concept applications demonstrate that the gas reporting method is a generalizable alternative to study microbial gene expression within soil where visual reporters are not compatible. I envision that this easy-to-use gas reporting method would facilitate the development of more sophisticated genetic circuits for applications in Earth, environmental, and planetary science