About this Biochemistry, Genetics and Molecular Biology article
Insights Into Receptor Structure and Dynamics at the Surface of Living Cells by Frederik Steiert; Peter Schultz; Siegfried Höfinger; Thomas D. Müller; Petra Schwille; Thomas Weidemann is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.
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
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- Author
- Frederik Steiert; Peter Schultz; Siegfried Höfinger; Thomas D. Müller; Petra Schwille; Thomas Weidemann
- Publisher
- Springer Science and Business Media LLC
- Published
- 2023
- Language
- EN
- Field
- Biochemistry, Genetics and Molecular Biology (Life Sciences)