Probing the Conformational States of Neurotensin Receptor 1 Variants by NMR Site-Directed Methyl Labeling.
2020
G-protein coupled receptors are key players mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Thus, various approaches have been developed in recent years to remedy this situation, including protein stabilization methods. This approach typically results in GPCRs that contain a varying number of mutations. Probing the functionality of such variants by in vitro and in vivo assays is often time-consuming and does not provide information on the conformational states that occur during activation. NMR spectroscopy has been proven to be a suitable technique to probe the conformational landscape of GPCRs in solution. Here, by using chemical modification of cysteine residues with an isotope-labeled methyl probe, we show that the conformational states of stabilized neurotensin receptor 1 variants can be efficiently assayed with 2D NMR experiments and correlated with their macroscopic activity profile. In addition, we show that the presented procedure can be utilized for the design of mutagenesis experiments to restore native-like conformational switching features in a stabilized receptor background. The presented chemical labeling approach will be broadly applicable in cases where low production yields do not permit uniform of amino-acid-selective labeling and where 1D NMR spectra of 19F-labeled receptors are not sufficient to resolve signal overlap.
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