1. β2-Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop 
Matthew T. Eddy, Tatiana Didenko, Raymond C. Stevens, Kurt Wüthrich 
Structure 
Volume 24, Issue 12, Pages (December 2016)
DOI: /j.str
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2. Structure 2016 24, 2190-2197DOI: (10.1016/j.str.2016.09.015)
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3. Figure 1
Locations of the 19F-NMR Probes in β2AR-T4L Used in This Work and Ligand-Efficacy-Dependent Effects on Local Conformational Equilibria Observed at C265
(A) Locations of TET 19F-NMR labels in the crystal structure of β2AR-T4L in complex with carazolol (PDB: 2RH1) (Cherezov et al., 2007). β2AR and T4L are shown as gray ribbon and blue surface representations, respectively. TET-labeled cysteines at positions 265, 327, and 341 are indicated as yellow spheres.
(B) 1D 19F-NMR spectra of β2AR-T4L(TETC265, C327S, C341A) and β2AR(TETC265, C327S, C341A) measured at 280 K for complexes with six different ligands and without addition of a ligand. The data for β2AR (TETC265, C327S, C341A) were taken from Liu et al. (2012a). Dashed vertical red and blue lines indicate the previously assigned chemical shifts (Liu et al., 2012a) of the active-like and inactive functional states for β2AR (TETC265, C327S, C341A), and NMR signals representing these states are shown in red (active-like) and blue (inactive). All experiments were measured at 280 K, and the following parameters were used to measure and process the spectra: data size 1,024 complex points, acquisition time 51 ms, 24,576 scans per experiment. Prior to Fourier transformation the data were multiplied with an exponential function with a line-broadening factor of 30 Hz and zero-filled to 2,048 points.
Structure  , DOI: ( /j.str )
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4. Figure 2
Helix VII Ligand-Efficacy-Related Conformational Equilibrium Is Preserved in β2AR-T4L Complexes Except for the Norepinephrine Complex
1D 19F-NMR spectra of β2AR-T4L(C265A, TETC327, C341A) and β2AR(C265A, TETC327, C341A) measured with six ligands and without addition of a ligand (“Apo”) are shown, as recorded at 280 K. The data for β2AR(C265A, TETC327, C341A) were taken from Liu et al. (2012a). Deconvolution with Lorentzian line shapes representing individual components are indicated in blue (inactive state) and red (active-like state). Dashed red and blue vertical lines correspond to the previously assigned chemical shifts for the active-like and inactive functional states for β2AR(C265A, TETC327, C341A) (Liu et al., 2012a). Thin black and thick gray lines indicate the experimental data and the fitting results, respectively. Chemical structures of the ligands used in the current work are displayed on the right, where the ethanolamine substituents are highlighted in yellow.
Structure  , DOI: ( /j.str )
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5. Figure 3
Visualizing the Effects of T4L on Local Conformational Equilibria Related to the G-Protein and Arrestin Signaling Pathways in β2AR-T4L
(A) Schematic side-view representation of β2AR. The transmembrane helices I to V are shaded in gray. The helices VI and VII, which are in equilibrium between two conformational states manifested by different NMR chemical shifts of 19F probes attached to C265 and C327 (Liu et al., 2012a), are represented as dashed rectangles in blue and cyan, respectively. The signaling pathways to G protein (blue) and β-arrestin (cyan) are indicated, and the positions of the 19F-NMR labels are shown by yellow spheres, where the active-like state is identified by a black circle. The orthosteric ligand binding cavity is indicated by green shading. For all ligands listed in (B), the response of the local conformational equilibria at the intracellular tips of helices VI and VII in β2AR is related with the efficacy of the ligand bound, i.e., the population of the active-like state is higher for ligands with higher efficacy (Liu et al., 2012a; see Figures 1 and 2).
(B) Schematic side views of β2AR-T4L. Same presentation as in (A), except that helix VI is represented by solid blue lines, the T4L fusion protein is depicted as a red oval, and its impact on local conformational equilibria is indicated by red arrows and the intensity of red shading.
(C) View from the extracellular surface of the β2AR-T4L complexes shown in (B). Circles indicate the helices I to VII. Ligands are represented in green, with black rectangles indicating hydrogen bonds between the ligand, N113 in helix III, and N312 in helix VII. In the upper panel, red arrows indicate that long-range effects from T4L cause a contraction of the ligand binding pocket (see text). Overall, (B) and (C) illustrate that the T4L fusion has a strong effect on the adjacent helix VI, and that a long-range effect via the orthosteric ligand binding cavity on the 19F-NMR probe at the tip of helix VII is suppressed in the presence of ligands with hydrophobic substituents at the ethanolamine end (Figure 2) (see text).
Structure  , DOI: ( /j.str )
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