Trimethylsilyl reporter groups for NMR studies of conformational changes in G protein‐coupled receptors

Abstract

Large membrane proteins such as G protein‐coupled receptors (GPCRs) are difficult for NMR study due to severe signal overlaps and unfavorable relaxation properties. We used a trimethylsilyl (TMS) group as a reporter group for 1H NMR study of conformational changes in proteins, utilizing high‐intensity 1H NMR signals near 0 p.p.m. The β2‐adrenergic receptor was labeled with TMS groups at two cysteines located at the cytoplasmic ends of helices VI and VII. Binding of various ligands led to changes in 1H NMR signals, which manifested that helix VI is sensitive to G protein‐specific activation, whereas helix VII is sensitive to β‐arrestin‐specific activation. Thus, the TMS group is a useful reporter group in NMR for studying conformational changes in membrane proteins such as GPCRs.

Site-specific DNA functionalization through the tetrazene-forming reaction in ionic liquids

“…S16 †), likely due to the competing Staudinger reduction pathway of the reaction between phosphine and azide reagents. Moreover, NMR study demonstrated that treatment of benzylamine (reactive for the amine-azide coupling) or aniline (unreactive) compounds with an alkylazide reagent with triphenylphosphine did not show meaningful difference in 31 P NMR with regard to PPh 3 and O]PPh 3 amounts (Fig. S17 †), further supporting our mechanistic hypothesis that the coupling reaction does not generate triphenylphosphine oxide and that signicant amount of the phosphine/azide reagents also would undergo the Staudinger reduction process.…”

“…The trimethylsilyl (TMS) group has been used increasingly as a chemical reporter in structural biology research because of its characteristic chemical shi in nuclear magnetic resonance (NMR) spectra. 30,31 Despite the relatively large steric bulk of the trimethylsilyl group, the TMS-methylazide (1b) could be used to introduce the TMS group to the DNA. Tertiary amine-containing reagents such as 1c or 1d did not decrease the efficiency of the modication, and could be useful for introduction of an additional positive charge to the biomolecule.…”

Site-Specific DNA Functionalization through the Tetrazene-Forming Reaction in Ionic Liquids

“…The trimethyl-silyl (TMS) group has been used increasingly as a chemical reporter in structural biology research because of its characteristic chemical shift in nuclear magnetic resonance (NMR) spectra. 26,27 Despite the relatively large steric bulk of the trimethylsilyl group, the TMS-methylazide (1b) could be used to introduce the TMS group to the DNA. Tertiary amine-containing reagents such as 1c or 1d did not decrease the efficiency of the modification, and could be useful for introduction of an additional positive charge to the biomolecule.…”

Dynamic G Protein-Coupled Receptor Signaling Probed by Solution NMR Spectroscopy

“…(a) Various intrinsic and extrinsic NMR probes can be incorporated into GPCRs. Multiple conformations of GPCR are observed in (b) 1D 19 F and 1 H NMR spectra (left spectrum adapted with permission from ref , copyright 2018 National Academy of Sciences; right spectrum adapted with permission from ref , copyright 2019 Federation of European Biochemical Societies), (c) 2D 1 H– 15 N NMR spectra (left spectrum adapted with permission from ref , copyright 2007 Springer Nature Ltd.; right spectrum adapted with permission from ref , copyright 2016 Springer Nature Ltd.), and (d) 2D 1 H– 13 C NMR spectra (left spectrum adapted with permission from ref , copyright 2019 Elsevier; right spectrum adapted with permission from ref , copyright 2012 Springer Nature Ltd.). (e) Correlations between chemical shifts and GPCR function.…”