Insights into receptor structure and dynamics at the surface of living cells

Abstract

Evaluating protein structures in living cells remains a challenge. Here, we investigate Interleukin-4 receptor alpha (IL-4Rα) into which the non-canonical amino acid bicyclo[6.1.0]nonyne-lysine (BCNK) is incorporated by genetic code expansion. Bioorthogonal click labeling is performed with tetrazine-conjugated dyes. To quantify the reaction yield in situ, we develop brightness-calibrated ratiometric imaging, a protocol where fluorescent signals in confocal multi-color images are ascribed to local concentrations. Screening receptor mutants bearing BCNK in the extracellular domain uncovered site-specific variations of both click efficiency and Interleukin-4 binding affinity, indicating subtle well-defined structural perturbations. Molecular dynamics and continuum electrostatics calculations suggest solvent polarization to determine site-specific variations of BCNK reactivity. Strikingly, signatures of differential click efficiency, measured for IL-4Rα in ligand-bound and free form, mirror sub-angstrom deformations of the protein backbone at corresponding locations. Thus, click efficiency by itself represents a remarkably informative readout linked to protein structure and dynamics in the native plasma membrane.

Cannabinoid Receptor Signaling is Dependent on Sub-Cellular Location

“…This proved to be an efficient method for tagging the receptor at the plasma membrane while having little impact on CB1R signaling and trafficking, and we identified sites for efficient incorporation into both CB1R and CB2R. Previously, other groups have employed genetic code expansion to study GPCRs and other membrane receptors in a variety of settings (33, 51). We believe our SPIEDAC incorporation site will allow for high flexibility in the choice of dye and will hence provide a valuable tool for future studies of CB1R in intact cells.…”

“…Previously, other groups have employed genetic code expansion to study GPCRs and other membrane receptors in a variety of settings (33,51). We believe our SPIEDAC incorporation site will allow for high flexibility in the choice of dye and will hence provide a valuable tool for future studies of CB1R in intact cells.…”

Bioorthogonal Reactions in Bioimaging

“…Furthermore, click-tagged fluorescent receptors can also be applied to study receptor domain dynamics using bioluminescence resonance energy transfer (BRET) [ 125 ]. Using BCN-Lys mediated labeling by Weidemann et al provided insights into interleukin-4 receptor alpha (IL-4Rα) structure and dynamics by screening receptor mutants bearing BCN-Lys in the extracellular domain [ 126 ]. Brightness-calibrated ratiometric imaging revealed site-specific variations of both labeling efficiency and IL-4 binding affinity and allowed detection of sub-angstrom shifts of receptor conformations in their native environment.…”

Enzyme-Mediated Bioorthogonal Cascade Catalytic Reaction for Metabolism Intervention and Enhanced Ferroptosis on Neuroblastoma

“…Bioorthogonal chemistry has carried out many non-natural chemical reactions/transformations in complex living systems without interfering with the native bioprocesses, serving as a powerful toolkit to understand biological processes as well as fight against diseases. − As an effective complement to chemical biology, bioorthogonal catalysis facilitates in situ imaging, regulation, and therapeutic applications. ,,− Abiotic transition metal catalysts (TMCs), including copper, iron, platinum, iridium, and ruthenium, have been demonstrated as outstanding candidates for implementing bioorthogonal chemistry. − However, unexpected toxicity, limited catalytic activity, and low stability have substantially hampered the application of TMCs in complex biological environments. , Moreover, proteins, metabolites, and signal molecules in living organisms are highly ordered, and their functions are precisely regulated in space-time. In terms of accuracy, sensitivity, and specificity, the catalytic behavior of artificial chemosynthetic TMCs is much lower than that of natural biological processes.…”