Insights into receptor structure and dynamics at the surface of living cells
F. Steiert, P. Schultz, S. Hofinger, T. Muller, P. Schwille, and T. Weidemann. Nat Commun, 14 (1):
1596(2023)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..
DOI: 10.1038/s41467-023-37284-4
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 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.
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.
%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
}