Browsing by Author "Sadler, Fredrik"
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Item Autoregulation Of G Protein-Coupled Receptor Signaling Through The Third Intracellular Loop(2023-04) Sadler, FredrikThe 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.Item Development of Methods and Tools for Cell-Free Synthetic Biology Applications(2017-08) Sadler, FredrikA 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.