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Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes

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ABSTRACT

The expression, functional reconstitution and first NMR characterization of the human growth hormone secretagogue (GHS) receptor reconstituted into either DMPC or POPC membranes is described. The receptor was expressed in E. coli. refolded, and reconstituted into bilayer membranes. The molecule was characterized by 15N and 13C solid-state NMR spectroscopy in the absence and in the presence of its natural agonist ghrelin or an inverse agonist. Static 15N NMR spectra of the uniformly labeled receptor are indicative of axially symmetric rotational diffusion of the G protein-coupled receptor in the membrane. In addition, about 25% of the 15N sites undergo large amplitude motions giving rise to very narrow spectral components. For an initial quantitative assessment of the receptor mobility, 1H-13C dipolar coupling values, which are scaled by molecular motions, were determined quantitatively. From these values, average order parameters, reporting the motional amplitudes of the individual receptor segments can be derived. Average backbone order parameters were determined with values between 0.56 and 0.69, corresponding to average motional amplitudes of 40–50° of these segments. Differences between the receptor dynamics in DMPC or POPC membranes were within experimental error. Furthermore, agonist or inverse agonist binding only insignificantly influenced the average molecular dynamics of the receptor.

No MeSH data available.


1H-13C NMR order parameters for backbone and side-chain carbons of the uniformly 13C labeled GHS receptor reconstituted into POPC (filled symbols) and DMPC membranes (open symbols) at varying CP contact times and a temperature of 37 °C.C-H dipolar coupling constants were measured using DipShift experiments and averaged over several signals in the same 13C chemical shift region or for specific peaks.
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f4: 1H-13C NMR order parameters for backbone and side-chain carbons of the uniformly 13C labeled GHS receptor reconstituted into POPC (filled symbols) and DMPC membranes (open symbols) at varying CP contact times and a temperature of 37 °C.C-H dipolar coupling constants were measured using DipShift experiments and averaged over several signals in the same 13C chemical shift region or for specific peaks.

Mentions: The average order parameters of the resolved segments of the GHS receptor in POPC and DMPC membranes are reported in Fig. 4. Similar to the static 15N measurements, 1H-13C DipShift experiments that use a cross polarization transfer step are biased by the presence of motions in the molecule. At short CP contact time, only the rigid segments are efficiently excited and contribute to the spectral intensity, which most likely involve the transmembrane helices of the receptor. DipShift experiments acquired at longer CP contact times also excite the more mobile parts of the receptor, which are constituted by larger loops and tail regions. All 13C sites are equally excited if directly excited DipShift experiments are carried out. As a consequence, the measured order parameters are largest for short CP contact times and decrease upon increase of the contact times. Lowest order parameters are always determined for the directly excited DipShift experiments. In addition to the contact time dependence of the order parameters, we also observe order parameter differences for the individual positions in the protein backbone and sidechain. Highest order parameters are observed for the Cα and Gly Cα sites, followed by CH2 groups in the side chain and lowest order parameters are observed for the CH3 groups. Order parameters of the GHS receptor in POPC membranes are typically slightly higher than those in DMPC bilayers but the difference is much smaller as determined for the Y2 receptor before2324. Without the motional bias of the CP experiment, lowest order parameters determined for the protein backbone were 0.60–0.69 for POPC and 0.56–0.65 for DMPC membranes. These values are slightly higher than what has been observed for the Y2 receptor before2324. Order parameters values of the GHS receptor in DMPC and POPC membranes are reported in Tables S1 and S2 in the Supporting Information.


Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes
1H-13C NMR order parameters for backbone and side-chain carbons of the uniformly 13C labeled GHS receptor reconstituted into POPC (filled symbols) and DMPC membranes (open symbols) at varying CP contact times and a temperature of 37 °C.C-H dipolar coupling constants were measured using DipShift experiments and averaged over several signals in the same 13C chemical shift region or for specific peaks.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384189&req=5

f4: 1H-13C NMR order parameters for backbone and side-chain carbons of the uniformly 13C labeled GHS receptor reconstituted into POPC (filled symbols) and DMPC membranes (open symbols) at varying CP contact times and a temperature of 37 °C.C-H dipolar coupling constants were measured using DipShift experiments and averaged over several signals in the same 13C chemical shift region or for specific peaks.
Mentions: The average order parameters of the resolved segments of the GHS receptor in POPC and DMPC membranes are reported in Fig. 4. Similar to the static 15N measurements, 1H-13C DipShift experiments that use a cross polarization transfer step are biased by the presence of motions in the molecule. At short CP contact time, only the rigid segments are efficiently excited and contribute to the spectral intensity, which most likely involve the transmembrane helices of the receptor. DipShift experiments acquired at longer CP contact times also excite the more mobile parts of the receptor, which are constituted by larger loops and tail regions. All 13C sites are equally excited if directly excited DipShift experiments are carried out. As a consequence, the measured order parameters are largest for short CP contact times and decrease upon increase of the contact times. Lowest order parameters are always determined for the directly excited DipShift experiments. In addition to the contact time dependence of the order parameters, we also observe order parameter differences for the individual positions in the protein backbone and sidechain. Highest order parameters are observed for the Cα and Gly Cα sites, followed by CH2 groups in the side chain and lowest order parameters are observed for the CH3 groups. Order parameters of the GHS receptor in POPC membranes are typically slightly higher than those in DMPC bilayers but the difference is much smaller as determined for the Y2 receptor before2324. Without the motional bias of the CP experiment, lowest order parameters determined for the protein backbone were 0.60–0.69 for POPC and 0.56–0.65 for DMPC membranes. These values are slightly higher than what has been observed for the Y2 receptor before2324. Order parameters values of the GHS receptor in DMPC and POPC membranes are reported in Tables S1 and S2 in the Supporting Information.

View Article: PubMed Central - PubMed

ABSTRACT

The expression, functional reconstitution and first NMR characterization of the human growth hormone secretagogue (GHS) receptor reconstituted into either DMPC or POPC membranes is described. The receptor was expressed in E. coli. refolded, and reconstituted into bilayer membranes. The molecule was characterized by 15N and 13C solid-state NMR spectroscopy in the absence and in the presence of its natural agonist ghrelin or an inverse agonist. Static 15N NMR spectra of the uniformly labeled receptor are indicative of axially symmetric rotational diffusion of the G protein-coupled receptor in the membrane. In addition, about 25% of the 15N sites undergo large amplitude motions giving rise to very narrow spectral components. For an initial quantitative assessment of the receptor mobility, 1H-13C dipolar coupling values, which are scaled by molecular motions, were determined quantitatively. From these values, average order parameters, reporting the motional amplitudes of the individual receptor segments can be derived. Average backbone order parameters were determined with values between 0.56 and 0.69, corresponding to average motional amplitudes of 40–50° of these segments. Differences between the receptor dynamics in DMPC or POPC membranes were within experimental error. Furthermore, agonist or inverse agonist binding only insignificantly influenced the average molecular dynamics of the receptor.

No MeSH data available.