Insights into receptor structure and dynamics at the surface of living cells (original) (raw)

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 multicolor 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 welldefined 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 subangstrom 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. Site-specific manipulation of proteins by genetic code expansion (GCE), a technique that introduces non-canonical amino acids (ncAA) into the otherwise native polypeptide chain, offers fascinating possibilities for the investigation of protein function in living cells 1,2. The introduced ncAA carries bioorthogonal reactivity for the site-specific conjugation of biophysical probes featuring negligible interference with components of the cell 3,4. A variety of chemical strategies termed click labeling emerged for site-specific conjugation of sensor molecules to ncAA under physiological conditions 5. As an alternative to metal-catalyzed click reactions, inverse electron-demand Diels-Alder cycloaddition (iEDDAC) of tetrazine derivatives to ncAA side chains presenting reactive dienophiles provides superior reaction rates for fast coupling to targets expressed via GCE 6-11. Bioorthogonal click labeling with fluorescent dyes was successfully combined with biophysical single-cell methods to address common issues in receptor research, like diffusion, oligomerization states, and conformational changes 12-18. However, only few attempts have been made to systematically quantify the yields of bioorthogonal reactions per se 19. Because the experimental throughput is limited, efforts to optimize click reaction conditions can be substantial 20,21. Fluorescence fluctuation analysis provides a simple means for transforming confocal images into molecular concentration maps 22 and is therefore a powerful extension for live cell imaging 23,24. Consequently, fluorescence fluctuation analysis was introduced as a routine validation protocol in bioorthogonal labeling 25. However, even elaborate instrumentation cannot circumvent the systematic errors inherent to single-cell measurements. Cells are far-from-equilibrium systems that carry out a myriad of processes on various time scales. Data curation must be carefully executed post-measurement to extract meaningful information. To mitigate these efforts, we here develop

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