%0 Journal Article %1 noauthororeditor %A Steiert, Frederik %A Schultz, Peter %A Hoefinger, Siegfried %A Mueller, Thomas D. %A Schwille, Petra %A Weidemann, Thomas %D 2023 %J Nature Communications %K own tdmueller %N 1596 %P 15 %R doi: 10.1038/s41467-023-37284-4 %T Insights into receptor structure and dynamics at the surface of living cells %U https://www.nature.com/articles/s41467-023-37284-4 %V 14 %X 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 bicyclo6.1.0nonyne-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.

@article{noauthororeditor, 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.}, added-at = {2023-06-20T12:46:45.000+0200}, author = {Steiert, Frederik and Schultz, Peter and Hoefinger, Siegfried and Mueller, Thomas D. and Schwille, Petra and Weidemann, Thomas}, biburl = {https://www.bibsonomy.org/bibtex/26fc294c0d94d627812b9be43849e4471/tdmueller}, doi = {doi: 10.1038/s41467-023-37284-4}, interhash = {00bce2e4e7ffcf3536bd71ec0d4fca4d}, intrahash = {6fc294c0d94d627812b9be43849e4471}, journal = {Nature Communications}, keywords = {own tdmueller}, month = {March}, number = 1596, pages = 15, timestamp = {2023-06-20T12:46:45.000+0200}, title = {Insights into receptor structure and dynamics at the surface of living cells}, url = {https://www.nature.com/articles/s41467-023-37284-4}, volume = 14, year = 2023 }