R-3 Repository :: Browsing by Author "Silberg, Jonathan J"

Browsing by Author "Silberg, Jonathan J"

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Characterizing the tolerance of near infrared fluorescent bacterial phytochromes to random backbone fission and circular permutation
(2016-04-25) Pandey, Naresh; Silberg, Jonathan J
Protein fission, fusion, and circular permutation have been used to convert green fluorescent protein (GFP) family members into biosensors that dynamically report on cellular processes, ranging from protein expression and metabolite concentrations to protein solubility, protein-protein interactions, and ligand-binding. Unfortunately, GFP are unsuitable for deep tissue reporting in animal models because the wavelengths of light used with these reporters is highly absorbed by tissues. In contrast, near infrared fluorescent protein (IFP and iRFP) reporters derived from bacterial phytochrome proteins (BphP) are excited by light in the near-infrared spectrum (~700 nm, less absorptive) and are better suited for probing cellular processes within tissues. IFP and iRFP can report on biological processes under anaerobic conditions because it uses biliverdin (BV) as a chromophore and does not require oxygen for maturation, a requisite for GFP maturation. Unlike GFP, IFP and iRFP are not yet able to report on wide-range of biological processes beyond gene expression. To report on gene expression, BphP must interlace its Per/ARNT/Sim (PAS) and cGMP phosphodiesterase/adenylcyclase/FhlA (GAF) domains into a topological knot. The extent to which this complex topology tolerates mutations (fission, fusion, and circular permutation) used to convert proteins into biosensors is not known. To better understand the tolerance of BphP to these types of mutational lesions, I have subjected IFP to random backbone fragmentation and iRFP to circular permutation using transposase mutagenesis. Screening a library of split IFP for fluorescent variants yielded thirteen unique fragmented IFP and with parent like spectral properties. These two-fragment IFP all required assistance from associating proteins for maximal fluorescence. These split sites displayed AND gate logic behavior when the ORFs encoding the different fragment are placed under distinct transcriptional regulation. In addition, screening a library of circularly permuted iRFP led to the discovery of twenty seven permuted iRFP variants with near infrared fluorescence. These variants arose from backbone fission in both the PAS and GAF domains, although the brightest permuted iRFP initiated at residues near the domain linker and termini. Biochemical analysis revealed that permuted iRFP display similar oligomerizatoin, quantum yield, and stability as native iRFP. These proteins also retained sufficient BV affinity serve as reporters of gene expression in mammalian cells without the addition of exogenous BV. The results described in this thesis represent the first study to map the tolerance of a BphP to random fragmentation and circular permutation. These results demonstrate that knotted BphP retain the ability to fold as their contact order changes, suggesting that these proteins can be further developed as reporters of biological processes like GFP. The split IFP represent a suite of assays that will be useful for monitoring the dynamics of a protein-protein interactions under conditions where split GFP do not yield strong signals. These split IFP can also be used to report on protein-ligand interactions that regulate protein oligomerization. The permuted iRFP should be useful for building molecular switches through domain insertion, in which the set of permuted iRFP is randomly inserted into other protein domains to couple the ligand binding to iRFP fluorescence.
Developing assays to study and screen engineered Escherichia coli NADPH-dependent sulfite reductase
(2024-04-19) Padron, Andrea; Silberg, Jonathan J
Living, cell-based bioelectronic devices are an emerging technology with broad applications in sensing, production, and remediation. These biotic-abiotic hybrids leverage one of biology’s key processes: electron transfer within and across the cellular membrane. As our understanding of electron transfer pathways expand, our ability to construct synthetic electron transfer pathways and engineer control over these reactions enable the creation of more efficient cell-material devices. One way to program electron transfer in microbes is to diversify the proteins that participate in these reactions (e.g., oxidoreductases) through protein engineering. However, successful enzyme engineering studies depend on appropriate assays to evaluate and characterize the desired variants. The Escherichia coli NADPH-dependent sulfite reductase is a heterododecameric complex that mediates one of the three known six-electron reductions in biological systems, catalyzing the reduction of sulfite to sulfide. Its higher order structure consists of an oxidative flavoprotein subunit and a reductase hemoprotein subunit that assemble in a unique asymmetrical stoichiometry to efficiently coordinate the large-volume electron transfer reaction. This oxidoreductase lends itself to numerous design opportunities due to (i) available crystal structures allowing rational design and (ii) essential catalytic activity that is crucial to E. coli metabolism in auxotrophic conditions, facilitating large-scale mutagenic library evaluation through a high-throughput growth selection. Herein, I describe my efforts in developing robust assays for studying engineered E. coli sulfite reductases. To enable recombinant expression of homogeneous protein, I modified an existing E. coli strain through a genetic deletion. I demonstrate that wild-type sulfite reductase can be easily purified in this strain by an affinity tag on the flavoprotein component without impacting oligomerization or catalytic function. I show that sulfite reductase activity can be monitored in vitro using an electrode and that this method presents an advantage over the traditional method when assessing activity in mutants. Additionally, I optimized a microplate-based endpoint assay that can be theoretically used to screen sulfite reductase mutants designed to accept electrons from other redox molecules beyond the native cofactor. This work supports future studies involving engineered E. coli sulfite reductase that may lead to the creation of new electron transfer biocomponents applicable to living bioelectronics.
Discovering and Calibrating Design Rules for Programming Adeno-Associated Virus Nanoparticles
(2015-12-03) Ho, Michelle Liane; Suh, Junghae; Silberg, Jonathan J; Tabor, Jeffrey J
Effective gene therapy must deliver therapeutic genes to disease sites while avoiding healthy tissue. However, engineering targeted gene delivery vectors to ensure exclusive delivery to diseased sites remains a challenge. Adeno-associated virus (AAV) is receiving increasing attention for its potential as a gene delivery vehicle because it offers several advantages: it is considered the safest viral vector, it infects human cells efficiently, and it can be genetically altered to improve therapeutic efficacy. However, even slight modifications to the virus capsid (the outer protein shell covering its genome) lead to unpredictable outcomes. Thus, a governing set of design rules for virus capsid assembly and function is needed to improve future engineering efforts. To this end, this thesis uncovers some of these rules by applying a computational model, often used in protein engineering, to the AAV capsid. A new strategy to improve AAV targeting was also explored by engineering AAVs to sense and become activated by extracellular proteases found in diseased tissues. The specificity of these protease-activatable viruses can be tuned to recognize a variety of protease profiles to treat a multitude of diseases. Design rules for these platform technologies are unveiled through their development and in-depth characterization. We also explore new motifs in the AAV capsid to further our understanding of AAV basic biology. Ultimately, these studies advance our ability to program virus nanoparticles for many biomedical applications.
Engineering multi-input gene regulation for applications in Synthetic Biology
(2015-04-17) Shis, David Liu; Bennett, Matthew R; Shamoo, Yousif; Silberg, Jonathan J; Tabor, Jeffrey J
Synthetic biology offers insight into molecular biology through the design and implementation of synthetic gene networks. One challenge in this effort is implementing transcriptional logic gates that enable synthetic gene networks to make decisions based on multiple inputs. However, the ability to implement transcriptional logic gates is inhibited by a lack of parts available to build them. In this work, we explore strategies for facilitating multi-input gene regulation in prokaryotes. That is, we develop methods for making the expression of a reporter gene dependent on two or more inputs in Escherichia coli. We first demonstrate how fragmentation of T7 RNA Polymerase (T7 RNAP) creates a multi-fragment transcription complex that facilitates AND transcriptional logic. We find split T7 RNAP to be functional in vivo and that both fragments of the split protein must be present for transcription from the T7 Promoter, PT7, to occur. We also find that the specificity of the split protein can be modified to create split protein mutants with orthogonal specificity. In addition to split T7 RNAP, we test the AND transcriptional logic made possible by co-expressing multiple chimeric LacI/GalR transcriptional repressors. We find that each chimeric repressor regulates the operator site of its DNA binding domain (DBD) according to the ligand sensed by its ligand binding domain(LBD). By co-expressing multiple chimeric repressors, we find each repressor independently regulates its DBD's operator. As a result, the number of inputs at a promoter relates directly to the number of species of chimeric repressors with the same DBD. Further, by modifying the DBD we find that we can create chimeras with orthogonal specificities that facilitate an orthogonal open reading frame. We find expression of our chimeric repressors en mass facilitates regulation such as a four-input transcriptional AND gate or two orthogonal transcriptional AND gates. Split T7 RNAP and the coexpression of chimeric LacI/GalR repressors both demonstrate strategies for multi-input gene regulation in prokaryotes. This work also suggest strategies for the engineering of additional components for use in synthetic gene networks.
Examining how adenylate kinase orthologs differ in their tolerance to circular permutation
(2016-11-18) Jones, Alicia Michelle; Silberg, Jonathan J
In nature, protein sequence rearrangements can arise as proteins evolve through a process called circular permutation (CP). These rearrangements result in the covalent linkage of the original protein termini and the creation of new termini elsewhere in the protein backbone. Although the overall tertiary structure of a protein remains the same, CP can have a wide variety of effects on protein dynamics, stability, and activity. The use of CP in protein engineering has yielded proteins with improved catalytic activity, and it has also been valuable in building molecular switches using domain insertion of circularly permuted proteins. However, we cannot yet anticipate how proteins tolerate permutation, and current models are limited in their predictions. My thesis research investigates how adenylate kinase (AK), a well-studied phosphotransferase, tolerates CP. Using AK orthologs with a range of thermostabilities, I improved upon a method for creating combinatorial libraries of circularly permuted AKs using transposase mutagenesis. Application of this method to a hyperthermophilic AK revealed that several structural metrics correlated with permutation tolerance, including: (i) the distance of the new protein termini to the catalytic site, (ii) the sequence diversity at the new termini within a multiple sequence alignment of bacterial AKs, and (iii) the structural deviation of the new termini in superimposed AK structures. In addition, I showed that a trade-off exists between consistently expressing permuted AK in a combinatorial library and minimizing N-terminal peptide additions. Subsequent studies explored AK tolerance to permutation in thermophilic, mesophilic, and psychrophilic species. Next generation sequencing was used to assess biases in permutation libraries and to mine these permuted libraries for functional AK. The method described for building combinatorial libraries using transposase mutagenesis will be applicable to any protein and will simplify studies of permutation tolerance across many proteins in parallel. The results of my thesis work also have implications for understanding protein tolerance to CP by providing insight into the structural parameters that correlate with retention of structure and function. Finally, comparisons of AK permutation tolerance in multiple orthologs will aid in the development of better models for predicting protein tolerance to permutation.
Post-Translational Control over Metabolic Labeling of Newly Synthesized Proteins
(2018-04-11) Thomas, Emily Elizabeth; Silberg, Jonathan J
A variety of aminoacyl-tRNA synthetases (aaRSs) have had their substrate specificities altered through protein engineering to create mutant aaRSs that can charge tRNA with noncanonical amino acids. While the activities of these mutant aaRSs can be dynamically controlled in cells using conditional promoters, our ability to quickly switch aaRS-mediated metabolic labeling on and off remains limited because we have not yet discovered ways to directly control aaRS activity post-translationally. I have engineered a first generation of ligand-responsive aaRSs by targeting Escherichia coli methionyl-tRNA synthetase (MetRS). A previously developed mutant, L13N/Y260L/H301L MetRS (NLL-MetRS), can charge tRNA with azidonorleucine (Anl), allowing bioorthogonal metabolic labeling. To develop NLL-MetRS switches, I used a combinatorial approach to generate libraries of split MetRS fused to different protein-protein interactions. I screened these libraries for active split MetRS using bacterial complementation and discovered six split MetRS whose fragments cooperatively function when fused to a pair of interacting proteins. I introduced the mutations necessary to change the specificity of MetRS from methionine to Anl and found that bacterial complementation in split MetRS correlates with metabolic labeling in split NLL-MetRS. I examined whether the activity of split NLL-MetRS can be regulated by either fusing the fragments to a pair of proteins whose interaction is stabilized by ligand binding or by fusing the fragments to the termini of a single protein domain that exhibits a ligand-dependent conformational change. When NLL-MetRS fragments were fused to FKBP12 and the FKBP-rapamycin binding domain of mTOR (FRB), metabolic labeling was significantly enhanced in growth medium containing rapamycin, which stabilizes the FKBP12-FRB complex. Similarly, fusion of MetRS fragments to the termini of the ligand-binding domain of the human estrogen receptor alpha yielded a protein whose metabolic labeling was significantly enhanced in the presence of 4-hydroxytamoxifen. These protein switches are expected to be useful for extending control over metabolic labeling with Anl. Furthermore, this approach can be applied to metabolic labeling with additional non-natural amino acids by extending the combinatorial design strategy to structurally-related aaRS. Ligand-responsive aaRSs are expected to have applications in production of protein biomaterials, proteomic studies, and protein pharmaceutical biosynthesis.

Browsing by Author "Silberg, Jonathan J"

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