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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.

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Summary of the order parameter measurements of the GHS receptor in the absence of ligand (filled bars) and in the presence of the agonist ghrelin (light filled bars) and the inverse agonist KbFwLK(Pam)-NH2 (empty bars) performed this study.Order parameters were determined from DipShift experiments conducted using CP excitation at a contact time of 700 μs (A,B) as well as by direct excitation (C,D). The GHS receptor was reconstituted into POPC (A,C) or DMPC membranes (B,D) and investigated at a temperature of 37 °C.
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f5: Summary of the order parameter measurements of the GHS receptor in the absence of ligand (filled bars) and in the presence of the agonist ghrelin (light filled bars) and the inverse agonist KbFwLK(Pam)-NH2 (empty bars) performed this study.Order parameters were determined from DipShift experiments conducted using CP excitation at a contact time of 700 μs (A,B) as well as by direct excitation (C,D). The GHS receptor was reconstituted into POPC (A,C) or DMPC membranes (B,D) and investigated at a temperature of 37 °C.

Mentions: Agonists and inverse agonists shift the equilibrium of active and inactive G protein-coupled receptor towards the active or inactive conformation, respectively. As the GHS receptor has been reported to display a ~50% constitutive activity3546, studying the molecular dynamics of the receptor in the presence of either agonist or inverse agonist represents an interesting topic8. Figure 5 shows the order parameters of the GHS receptor in the absence as well as in the presence of either the agonist ghrelin or the inverse agonist KbFwLK(Pam)-NH2 recorded in DipShift experiments that were excited by CP at a contact time of 700 μs (A, B) or by direct 13C excitation (C, D) both in POPC (A, C) and DMPC membranes (B, D). Order parameters determined by CP at a contact time of 700 μs are generally higher than those obtained from directly excited DipShift experiments irrespective of the presence or absence of any ligand. Order parameters determined from the CP based DipShift experiments at 700 μs contact time, which preferentially detects the more rigid structures show a tendency to be slightly higher for the ligand bound GHS receptor. However, this is just a trend, which is not consistent for all receptor segments studied and only in a few cases outside of the error margin of the experiment, which was determined from preparations and measurements carried out in duplicate. Order parameters determined by direct 13C excitation, which do not have a bias by molecular motions with varying amplitudes are identical within the experimental error for all three studied cases. No systematic differences between receptor preparations in saturated DMPC versus monounsaturated POPC membranes were observed. All order parameters are reported in Tables S1 and S2.


Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes
Summary of the order parameter measurements of the GHS receptor in the absence of ligand (filled bars) and in the presence of the agonist ghrelin (light filled bars) and the inverse agonist KbFwLK(Pam)-NH2 (empty bars) performed this study.Order parameters were determined from DipShift experiments conducted using CP excitation at a contact time of 700 μs (A,B) as well as by direct excitation (C,D). The GHS receptor was reconstituted into POPC (A,C) or DMPC membranes (B,D) and investigated at a temperature of 37 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384189&req=5

f5: Summary of the order parameter measurements of the GHS receptor in the absence of ligand (filled bars) and in the presence of the agonist ghrelin (light filled bars) and the inverse agonist KbFwLK(Pam)-NH2 (empty bars) performed this study.Order parameters were determined from DipShift experiments conducted using CP excitation at a contact time of 700 μs (A,B) as well as by direct excitation (C,D). The GHS receptor was reconstituted into POPC (A,C) or DMPC membranes (B,D) and investigated at a temperature of 37 °C.
Mentions: Agonists and inverse agonists shift the equilibrium of active and inactive G protein-coupled receptor towards the active or inactive conformation, respectively. As the GHS receptor has been reported to display a ~50% constitutive activity3546, studying the molecular dynamics of the receptor in the presence of either agonist or inverse agonist represents an interesting topic8. Figure 5 shows the order parameters of the GHS receptor in the absence as well as in the presence of either the agonist ghrelin or the inverse agonist KbFwLK(Pam)-NH2 recorded in DipShift experiments that were excited by CP at a contact time of 700 μs (A, B) or by direct 13C excitation (C, D) both in POPC (A, C) and DMPC membranes (B, D). Order parameters determined by CP at a contact time of 700 μs are generally higher than those obtained from directly excited DipShift experiments irrespective of the presence or absence of any ligand. Order parameters determined from the CP based DipShift experiments at 700 μs contact time, which preferentially detects the more rigid structures show a tendency to be slightly higher for the ligand bound GHS receptor. However, this is just a trend, which is not consistent for all receptor segments studied and only in a few cases outside of the error margin of the experiment, which was determined from preparations and measurements carried out in duplicate. Order parameters determined by direct 13C excitation, which do not have a bias by molecular motions with varying amplitudes are identical within the experimental error for all three studied cases. No systematic differences between receptor preparations in saturated DMPC versus monounsaturated POPC membranes were observed. All order parameters are reported in Tables S1 and S2.

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.


Related in: MedlinePlus