%0 Journal Article %1 steiert2023insights %A Steiert, F. %A Schultz, P. %A Hofinger, S. %A Muller, T. D. %A Schwille, P. %A Weidemann, T. %D 2023 %J Nat Commun %K *Proteins/metabolism myOwn %N 1 %P 1596 %R 10.1038/s41467-023-37284-4 %T Insights into receptor structure and dynamics at the surface of living cells %U https://www.ncbi.nlm.nih.gov/pubmed/36949079 %V 14 %X Evaluating protein structures in living cells remains a challenge. Here, we investigate Interleukin-4 receptor alpha (IL-4Ralpha) 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-4Ralpha 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{steiert2023insights, abstract = {Evaluating protein structures in living cells remains a challenge. Here, we investigate Interleukin-4 receptor alpha (IL-4Ralpha) 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-4Ralpha 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 = {2024-02-15T15:08:22.000+0100}, author = {Steiert, F. and Schultz, P. and Hofinger, S. and Muller, T. D. and Schwille, P. and Weidemann, T.}, biburl = {https://www.bibsonomy.org/bibtex/2f090de982d8fdd315cc142b71de2f8ec/jvsi_all}, doi = {10.1038/s41467-023-37284-4}, interhash = {c8db1121756c5650a80b42ee6ac87d30}, intrahash = {f090de982d8fdd315cc142b71de2f8ec}, issn = {2041-1723 (Electronic) 2041-1723 (Linking)}, journal = {Nat Commun}, keywords = {*Proteins/metabolism myOwn}, note = {Steiert, Frederik Schultz, Peter Hofinger, Siegfried Muller, Thomas D Schwille, Petra Weidemann, Thomas eng Research Support, Non-U.S. Gov't England 2023/03/24 Nat Commun. 2023 Mar 22;14(1):1596. doi: 10.1038/s41467-023-37284-4.}, number = 1, pages = 1596, timestamp = {2024-02-15T15:08:22.000+0100}, title = {Insights into receptor structure and dynamics at the surface of living cells}, type = {Journal Article}, url = {https://www.ncbi.nlm.nih.gov/pubmed/36949079}, volume = 14, year = 2023 }