1. Volume 111, Issue 12, Pages 2620-2628 (December 2016)
Construction of Structural Mimetics of the Thyrotropin Receptor Intracellular Domain 
Olga Press, Tatiana Zvagelsky, Maria Vyazmensky, Gunnar Kleinau, Stanislav Engel 
Biophysical Journal 
Volume 111, Issue 12, Pages (December 2016)
DOI: /j.bpj
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2. Figure 1
The 6-Helix scaffold as a soluble mimetic of the GPCR TMD. (A) β2-AR/G-(s) complex crystal structure (PDB: 3SN6) (19). ICL-2 and ICL-3 are shown in pink, and Gα-(s) is shown as light purple ribbons. (B) The structure of the 6-Helix scaffold (33) reconstituted from the crystal structure of the HIV gp41 core (PDB: 1F23 (58)) is oriented so that side III faces the viewer. The 6-Helix helices used for insertion of the ICL-2 and ICL-3 elements of GPCR are colored according to their counterpart GPCR TM helices in (A). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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3. Figure 2
The 6-Helix side III TSHR ICD mimetics bind Gα-(s) more efficiently than the side II mimetics. Binding was measured by microscale thermophoresis after a 1 h incubation of 200 nM fluorescently labeled Gα-(s) at room temperature with increasing concentrations of unlabeled 6-Helix III-ICL2/3 (side III mimetic) or 6-Helix II-ICL2/3 (side II mimetic). The original 6-Helix scaffold protein, which was used as a negative control in the experiments, did not demonstrate any specific binding (these data were not plotted due to a low signal/noise ratio). The data were analyzed by the Prism 6 program (GraphPad) using a nonlineal regression and the standard slope sigmoidal function. Values presented are the means ± SD of three independent experiments performed in duplicates.
Biophysical Journal  , DOI: ( /j.bpj )
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4. Figure 3
The 6-Helix TSHR ICD mimetics stimulate Gα-(s)-catalyzed GTP hydrolysis. Recombinant Gα-(s) subunits (5 μM) were preincubated with increasing concentrations of 6-Helix TSHR ICD mimetics for 30 min at room temperature and the reaction was started by the addition of 300 μM GTP. After a 15 min incubation at 30°C, Pi liberated by the Gα-catalyzed GTPase reaction was measured calorimetrically using a PiColorLock reagent (Innova Biosciences). The data were analyzed by the Prism 6 program using the function V = (1 − x) × Vi + x × Vf, where Vi is the basal rate of Gα-catalyzed GTP hydrolysis, Vf is the rate of GTP hydrolysis catalyzed by the Gα/ICD-mimetic complex (at saturation), and x is the fraction of Gα subunit in the complex with ICD mimetics (x = [GM]/[Gαt]). Here, GM is the Gα/ICD-mimetic complex whose concentration was explicitly calculated from the equation ([Mt−GM]⋅[Gαt−GM])/[GM]=Kd, where Gαt and Mt are the total assay concentrations of the Gα subunits (5 μM) and ICD mimetics, respectively. The original 6-Helix scaffold protein used as a negative control in the experiments did not show any specific activation (not shown). Values presented are the means ± SD of three independent experiments performed in duplicates. The apparent Kd values calculated for Gα-(s) complexes with 6-Helix III-ICL2/3, 6-Helix III-ICL2, and 6-Helix III-ICL3 mimetics were 0.7, 9.6, and 11.5 μM, respectively.
Biophysical Journal  , DOI: ( /j.bpj )
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