Browsing by Subject "protein engineering"

Now showing 1 - 7 of 7
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    Autoregulation Of G Protein-Coupled Receptor Signaling Through The Third Intracellular Loop
    (2023-04) Sadler, Fredrik
    The third intracellular loop (ICL3) of the G protein-coupled receptor (GPCR) fold is important for the signal transduction process downstream of receptor activation. Despite this, ICL3’s lack of defined structure, combined with its high sequence divergence among GPCRs, obfuscates characterization of its involvement in receptor signaling. Previous studies focusing on the β2 adrenergic receptor (β2AR) suggest that ICL3 is involved in the structural process of receptor activation and signaling. We derive mechanistic insights into ICL3s role in β2AR signaling, finding that ICL3 autoregulates receptor activity through a dynamic conformational equilibrium between states that block or expose the receptor’s G protein binding site. We demonstrate the importance of this equilibrium for receptor pharmacology, finding that G protein-mimetic effectors bias ICL3’s exposed states to allosterically activate the receptor. Our findings additionally reveal that ICL3 tunes signaling specificity by inhibiting receptor coupling to G protein subtypes that weakly couple to the receptor. Despite the sequence diversity of ICL3, we demonstrate that this negative G protein selection mechanism through ICL3 extends to GPCRs across the superfamily, expanding upon the framework for how receptors mediate G protein subtype selective signaling. Furthermore, our collective findings motivate ICL3 as an allosteric site for receptor and signaling pathway specific ligands.
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    Constrained Diversification Enhances Protein Ligand Discovery and Evolution
    (2017-04) Woldring, Daniel
    Engineered proteins have strongly benefited the effectiveness and variety of precision drugs, molecular diagnostic agents, and fundamental research reagents. A growing demand for new therapeutics motivates the innovative use of natural proteins – improving upon their native properties – as well as discovering proteins with entirely new functionality. Importantly, these are fundamentally separate goals. While evolving improved function can result from making a few carefully chosen mutations, discovering novel function often requires giant leaps to be taken in protein sequence space. Discovering novel function is a notoriously challenging task. The immensity of sequence space (e.g. proteins of length n have 20^n unique options) makes it essentially impossible to experimentally or computationally test all possible protein sequences. Within this space, the landscape is incredibly barren and rugged (i.e. most sequences lack function entirely and making small changes to a protein often damage the activity). Rather than randomly mutating a protein, combinatorial protein libraries provide a systematic and efficient approach for searching sequence space. This method offers precise control over which protein sites are mutated and which amino acids are allowed at the diversified sites. To improve the likelihood of sampling useful sequences, numerous techniques can elucidate the structure-function relationships in proteins. Generally, these techniques have not been applied to combinatorial library design; however, we propose that some, or all, could be greatly beneficial in this area. In this thesis work, protein libraries are designed for the purpose of discovering high affinity, specific binders to a collection of interesting targets. High-throughput sequencing of evolved binders, natural protein-protein interface composition, structural assessment, and computational analysis of stability upon mutation collectively informed sitewise library designs – residues predicted to support function were allowed but destabilizing residues or those not likely to benefit function were avoided. We use multiple small protein scaffolds (affibody and fibronectin) as model systems to test the hypothesis that constrained sitewise diversity will improve the efficiency of novel protein discovery. This hypothesis was experimentally supported by a direct comparison of high-affinity ligand discovery between the sitewise constrained library and a uniformly diversified library (i.e. allowing all 20 residues at each diversified site). The constrained library showed a 13-fold improved likelihood of binder discovery. Moreover, the constrained library variants demonstrated superior thermal stability (Tm 15 °C higher than unbiased variants). This work provides further evidence that sitewise diversification of protein scaffolds can improve the overall quality of combinatorial libraries by offering broad coverage of sequence space without sacrificing stability.
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    Development of Methods and Tools for Cell-Free Synthetic Biology Applications
    (2017-08) Sadler, Fredrik
    A primary advantage of bead surface display is the potential for highly controlled, multivalent display of immobilized protein. To realize this potential, we built a bead surface display platform with multivalency in mind. Starting with a display particle with dense functional groups, we systematically designed and synthesized a bead saturated in DNA and protein attachment sites utilizing chemoselective coupling mechanisms. With the potential of tens of millions of biomolecule attachment sites, we optimized the biological steps of genotype and phenotype immobilization empirically. Starting with a single gene copy per bead to mimic monoclonality, we amplified genotype onto the bead surface to a sufficient degree to express attachable protein and, yet sparse to leave most of the bead surface area exposed for protein attachment. We systematically rescued the in vitro transcription step to maximize protein expression from the immobilized context. Our methodology enables more insensitive and indirect detection mechanisms for rank-sort screening of protein libraries.
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    Engineering Adeno-Associated Virus for Receptor-Mediated Gene Delivery
    (2021-03) Zdechlik, Alina
    Adeno-associated virus (AAV) is a useful gene delivery tool for clinical and basic research applications. Since its discovery nearly 60 years ago, it has become a popular vector because of its small size and low immunogenicity. However, natural tissue tropisms are limited and frequently not useful. Past work modifying the capsid has been limited by structural constraints and a lack of modularity. I aimed to address this problem by creating a modular retargeting system in which the AAV capsid can be rapidly retargeted to any given antigen without newly mutating capsid proteins. I characterized AAV-antibody composites produced by incorporating a small DNA binding domain into one of the AAV capsid proteins and using chemical conjugation to attach the paired DNA sequence to an antibody. I demonstrated that these antibody-AAV conjugates are capable of infecting cells via the antibody-antigen interaction in immortalized and primary cells. Additionally, I created six capsid variants incorporating small targeting scaffolds into each AAV capsid protein. These variants will enable future researchers to select variants from scaffold libraries in vivo, making more specific, better targeting moieties. Using these retargeted vectors, I worked with colleagues to target prostate tumors in vivo and to deliver new payloads with therapeutic potential. A collaborator had recently developed an antibody against a prostate tumor stromal marker, fibroblast activation protein alpha (FAP). By conjugating this antibody to my modified virus, we were able to deliver a fluorescent marker specifically to the tumor. Next, we plan to deliver therapeutic payloads to treat the tumor with minimal off-target effects. Additionally, I assisted in designing and testing a version of prime editor suitable for delivery by AAV. While the efficiency of this tool is still relatively low, it represents an important starting point for adaption of prime editor for use in vivo. In future work, we hope to use antibody-targeted AAV to deliver this and other editing reagents in murine disease models. Finally, to further investigate the landscape of the AAV capsid, we prepared a domain insertion library using a protocol recently developed by the lab. The results from this study will provide valuable information about the plasticity of the AAV capsid that can be used by future researchers to create new variants with added functionality.
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    Engineering Cell-Based Selections for Translatable Ligand Discovery
    (2021-12) Lown, Patrick
    Engineered protein ligands with specific, high-affinity binding to a biomarker that are differentially expressed in a disease state have been applied in a variety of therapeutic and diagnostic applications. Yeast surface display libraries coupled with high-throughput selection strategies have shown effectiveness in discovering and maturing ligands towards a variety of target molecules. These high-throughput selection strategies often require soluble protein as a target molecule. This requires that cell surface biomarkers with transmembrane domains, constituting a large class of interesting targets, be produced as recombinant extracellular domains due to the hydrophobic nature of the transmembrane domain. However, a variety of factors including poor stability, improper folding, incorrect post-translational modifications, the addition of chemical purification tags, and the lack of plasma membrane may result in additional non-natural epitopes or the masking of native epitopes. Thus, ligand discovery campaigns performed using recombinantly-produced extracellular domains may result in ligands that bind to the recombinant target but fail to recapitulate that binding towards full-length target on target-expressing cells or tissues. The use of either whole cells or detergent-solubilized cell lysate expressing full-length target has been successfully applied as an alternative to recombinant target in discovering ligands that translate binding to target-expressing cells and tissues in the context of cancer and blood-brain barrier targets. However, these selections lack the throughput to effectively screen full-sized yeast surface display libraries and are limited in their ability to select ligands from naïve libraries with limited affinity if overexpressing cell lines are not available. Finally, the heterogeneous nature of the mammalian cell surface often results in non target-specific ligands dominating the campaign, making the isolation of target-specific ligands difficult. All these factors limit the wide-spread use of cell-based selections. The work presented below aims to tackle each of these issues, as well as to elucidate the factors that affect successful cell-based selections and isolate panels of ligands with specific, high-affinity binding to biomarkers overexpressed in cancer. Naïve affibody and fibronectin libraries were sorted against cluster of differentiation 276 (CD276 or B7-H3) and cluster of differentiation 90 (CD90 or Thy1) by five selection strategies using recombinant extracellular domains and target-expressing cells. Cellular selection strategies provided a higher frequency of ligands that translate to binding on target-expressing cell monolayers, albeit with a relatively high degree of non target-specific binding. Sequential depletion on target-negative cell monolayers was insufficient to deplete these non target-specific binders, but pre-blocking yeast populations with disadhered target-negative cells provided significant depletion. Directed evolution through helix walking of a preliminary affibody molecule with modest but specific binding to CD276 (AC2, Kd = 310 ± 100 nM) resulted in a panel of CD276-specific ligands, including a sub-nanomolar binder (AC12, Kd = 0.9 ± 0.6 nM). Next, the use of mammalian cell-magnetic bead conjugates was investigated for use as effective cell-based pulldown agents to provide a new method of cell-based selection. This method displayed an order of magnitude higher throughput than traditional adherent cell panning, putting it on par with recombinant target magnetic-activated cell sorting (MACS), and was effective in enriching ligands under the same conditions as adherent cell panning in an EGFR model system, but failed to provide sufficient enrichment in a CD276 model system. Additionally, the use of an extended 641-amino acid linker was investigated to provide more consistent yeast-mammalian cell engagement and enhanced avidity. This extended linker provided enhanced enrichment in a >600-nM affinity ligand, 106 EGFR per cell system where the original 80-amino acid linker failed to provide effective enrichment (23 ± 7 vs. 0.8 ± 0.2, p = 0.004). This enrichment benefit was generalizable to a CD276 model system and mathematical modelling of the linkers as random chain polymers confirmed that this enhanced enrichment was likely due to the ability of an increased number of ligands to access the extracellular environment. Lastly, a method of high-throughput clonal specificity screening was developed using deep sequencing to observe clonal frequency in populations differentially panned on target-expressing and target-negative populations in the context of insulin-like growth factor receptor (IGF1R) and insulin receptor isoforms A (InsRA) and B (InsRB). Adherent cell panning yielded affibodies that were preferentially enriched on IGF1R-expressing cells relative to IGF1R-negative cells and affibodies and fibronectins that were preferentially enriched on InsR-expressing cells relative to parental HEK293T cells, but with limited isoform specificity. Deep sequencing of the IGF1R populations revealed several affibody sequences with specificity towards IGF1R-expressing cells. In total, the results contained in this thesis elucidate the factors that dictate successful cell-based panning and provide new methods to increase the throughput, enrichment, and specificity of cell-based panning to motivate wider adoption, as well as panels of compelling molecules with high-affinity, specific binding to cancer-relevant biomarkers for therapeutic and diagnostic applications.
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    HUH-endonuclease Mediated Protein-DNA Bioconjugation
    (2021-01) Tompkins, Kassidy
    Bioconjugation is a broad technique used to form direct linkages between and biomolecule and another functional chemical. More specifically, coupling functional proteins to programmable nucleic acids has enabled biotechnologies ranging from therapeutic diagnostics to super-resolution microscopy, indispensable for medical professionals and researchers alike. While current protein-nucleic acid bioconjugation methods have sprouted a myriad of biotechnologies, only a small number of compatible orthogonal, or non-cross-reactive, chemistries exist posing a bottleneck for multiplexing. In recent years, our research group has exploited HUH-endonucleases as fusion tags, dubbed HUH-tags, to create covalent adducts with specific single-stranded DNA (ssDNA) sequences. This dissertation addresses foundational biochemical knowledge and protein engineering vital for development of multiplexed HUH-tag applications. The ultimate goal of this research is to develop a toolkit of multiplexable HUH-tags ready for implementation into a wide variety of platforms. As a starting point, we explored the basis of ssDNA recognition of replication initiator proteins (Reps), which are a highly versatile version of HUH-tag that process DNA faster and are more compact than other HUH-endonucleases. Through analysis of co-crystal structures and development of a deep sequencing-based cleavage assay, HUH-seq, we demonstrate how Reps with subtle differences in specificity can be used as orthogonal HUH-tags and how specificity can be predictably engineered. Next, we characterized the cleavage kinetics of Reps and show how Reps from different families of organisms can reach completion in seconds. Finally, we show an example of rational Rep engineering by circular permutation, and how Reps can be exploited to form protein-RNA conjugates. The characterization and protein engineering described in this dissertation demonstrate how HUH-tags are a critical emerging bioconjugation technology.
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    Programmed mutagenesis and high-throughput methods to study protein recombination and epistasis
    (2021-04) Nedrud, David
    Protein science, which includes studying extant proteins and designing novel proteins, requires a fundamental understanding of protein properties and principles. Decades of research have discovered properties of protein structure, dynamics, and function. In this work, we build upon this research to study protein recombination and epistasis with high- throughput methods. First, we develop two methods for high-throughput screening, a deep mutational library generation method (SPINE), and an automated neuron profiling technique. SPINE improves both comprehensive and uniform coverage for deep mutational library generation, and our automated neuron profiling technique measures neuromodulation, developmental effect, and baseline shifts, which we use to develop a sodium channel modulator. Secondly, we use these methods to study over 300,000 recombined proteins and 648,000 pairwise amino acid substitutions. We show that the interaction between the inserted peptide and recipient protein regulates recombination fitness. Additionally, we show that negative epistasis is wide-spread yet restricted to proximal residues, and positive epistasis is predominantly long-range interactions and enriched in evolutionarily conserved, function-defining, and clade-specifying residues.