DOI: 10.1002/chem.201304928

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& Membrane Proteins

The G-Protein-Coupled Neuropeptide Y Receptor Type 2 is Highly Dynamic in Lipid Membranes as Revealed by Solid-State NMR Spectroscopy Peter Schmidt,[a] Lars Thomas,[a] Paul Mller,[a] Holger A. Scheidt,[a] and Daniel Huster*[a, b] Abstract: In spite of the recent success in crystallizing several G-protein-coupled receptors (GPCRs), a comprehensive biophysical characterization of these molecules under physiological conditions also requires the study of the molecular dynamics of these proteins. The molecular mobility of the human neuropeptide Y receptor type 2 reconstituted into dimyristoylphosphatidylcholine (DMPC) membranes was investigated by means of solid-state NMR spectroscopy. Static 15 N NMR spectra show that the receptor performs axially symmetric motions in the membrane, and several residues undergo large amplitude fluctuations. This was confirmed by quantitative measurements of the motional 1H,13C order parameter of the CH, CH2, and CH3 groups. In directly polarized

Introduction G protein-coupled receptors (GPCRs) are pharmacologically highly relevant membrane proteins that initiate signal transduction from external stimuli into the cell.[1] More than 800 GPCRs are found in the human genome, which all feature the same topology of seven transmembrane a-helices.[2] Until recently, structure information on these molecules was scarce, with only the crystal structure of bovine rhodopsin solved in the year 2000.[3] Since 2007, several crystal and one solid-state NMR spectroscopic structures of more than a dozen GPCRs have become available.[1, 2, 4] The fact that these important molecules have resisted crystallization for a long time is often ascribed to the loose structural arrangement of the heptahelical protein and the high molecular dynamics of GPCRs.[1] Indeed, crystallization only succeeds when the receptor is immobilized by anti- or nanobody binding, by removal of intracellular loops, or by thermostabilization through point mutations.[1, 5, 6] Despite the great success in the structural characterization of GPCRs, quantitative information on the dynamics of these [a] Dr. P. Schmidt, Dr. L. Thomas, P. Mller, Dr. H. A. Scheidt, Prof. Dr. D. Huster Institute of Medical Physics and Biophysics, University of Leipzig Hrtelstrasse 16–18, 04107 Leipzig (Germany) E-mail: [email protected] [b] Prof. Dr. D. Huster Department of Chemical Sciences TATA Institute of Fundamental Research Homi Bhaba Road, Mumbai, 400 005 (India) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201304928. Chem. Eur. J. 2014, 20, 4986 – 4992

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C NMR experiments, these order parameters showed astonishingly low values of SCH = 0.55, SCH2 = 0.33, and SCH3 = 0.17, which corresponds to segmental amplitudes of approximately 508 in the backbone and approximately 50–608 in the side chain. At physiological temperature, 2H NMR spectra of the deuterated receptor showed a narrow component that is indicative of molecular order parameters of S  0.3 superimposed with a very broad spectrum that could stem from the transmembrane a-helices. These results suggest that the crystal structures of GPCRs only represent a static snapshot of these highly mobile molecules, which undergo significant structural fluctuations with relatively large amplitudes in a liquid-crystalline membrane at physiological temperature.

molecules is typically only available from the crystallographic B factors.[7, 8] Analysis of the data for the available crystal structures reveals that much increased B factors are found for the interhelical loops and termini of the GPCRs.[7, 8] Furthermore, GPCR crystal structures often lack electron density for loop and tail structures, which suggests that these molecular segments are statically disordered or undergo large amplitude motions. For rhodopsin, several residues of the third intracellular loop and the C-terminal tail that extends from residue 324 are missing or poorly defined in the crystal structure owing to molecular mobility.[9] Direct quantitative information on GPCR mobility comes from site-specific electron paramagnetic resonance (EPR) measurements, which have demonstrated in detail the dynamic nature of rhodopsin.[8] The largest amplitude motions were found for the cytoplasmatic loops of the molecule as well as the C terminus.[10–14] The mobility of the R1 spin label attached to the transmembrane a-helices 1–3, 6, and 7 also increased upon activation of rhodopsin, thus indicating reduced packing in the core of the protein.[8, 14] Such conformational switching is the functionally most relevant dynamic mode of GPCRs.[10, 15] A second dynamic mode involves local fluctuations in the backbone and the side chain in thermodynamic equilibrium.[10, 15] These can occur as local fluctuations as well as correlated motions of entire secondary structure units. Although not directly involved in receptor function, such motions are believed to bring about the plasticity of the protein structure and facilitate larger conformational changes relevant for protein function.[15–17] In addition to EPR methods, NMR spectroscopy represents a powerful biophysical method to study protein dynam-

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Full Paper ics.[7, 17, 18] The timescale of motions covered by NMR spectroscopic methods ranges from tens of picoseconds to milliseconds and beyond.[15] Membrane proteins in a native lipid environment have to be studied by solid-state NMR spectroscopy,[19] which allows a more direct investigation of their dynamics because of the absence of the overall tumbling of soluble proteins.[20] Solid-state NMR spectroscopy can provide detailed information on the timescale and the specific geometry of molecular motions.[17, 21] Static solid-state NMR powder spectra are characterized by anisotropic interactions that cause large spectral widths. Any molecular dynamics analysis partially averages such interactions and causes a reduction in the width of the solid-state NMR spectra. If the molecular motion approaches isotropic reorientation, narrow lines as known from solution NMR spectroscopy are observed. Thus, the width of the solidstate NMR spectra contains information about the amplitude of motions provided the correlation time is shorter than the inverse of the spectral width. From the motionally averaged solid-state NMR powder spectrum, an order parameter can be derived, which varies between 1 (for completely rigid sites) and 0 (for isotropically mobile sites). In particular, such order parameters can be calculated from the 1H–13C dipolar or the 2 H–C quadrupolar coupling strengths. Intermediate timescale motions, which occur in the microsecond correlation time window in NMR spectroscopy, cause characteristic alterations of the solid-state NMR spectroscopic lineshape and can be described directly on the basis of the NMR spectra. Solid-state NMR spectroscopy has already provided important insights into GPCR structures. The largest body of data is available for rhodopsin, for which the ground and activated states have been characterized.[22–24] Also, the full structure of the chemokine receptor CXCR1 has recently been solved by solid-state NMR spectroscopy.[4] Furthermore, the conformations of the peptides neurotensin[25] and bradykinin[26] bound to their GPCRs have been determined by this method. It should be noticed that most of these studies have been carried out on frozen samples to study GPCR structure in the absence of molecular dynamics or to trap intermediates. At low temperature, most of the dynamics of GPCRs are lost. Herein, we have studied the molecular dynamics of the neuropeptide Y receptor type 2,[27] reconstituted into DMPC model membranes at several temperatures including physiological conditions. An astonishing molecular dynamics of the membrane-embedded GPCR was found. Although we cannot provide site-specific dynamics information for the receptor on account of resolution constraints, several dynamic modes of the molecule have been observed. Our results suggest that the Y2 receptor is highly mobile in the biologically active liquid-crystalline phase state of the membrane.

Results In our approach, we have prepared functional Y2 receptors from Escherichia coli inclusion bodies as shown for other receptors in the literature.[4, 28, 29] Functionality of the receptor in our preparations was shown by ligand-binding assays as published Chem. Eur. J. 2014, 20, 4986 – 4992

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Figure 1. Static 15N CP NMR spectra of uniformly 15N-labeled Y2 receptor in [D54]DMPC membranes at a temperature of A) 30 8C and B–D) 37 8C. In addition to temperature, the CP contact time varied between C) 70 ms, A, B) 2 ms, and D) 8 ms. Assignment of the prominent side chains is given in the spectrum acquired at the longest CP contact time.

before,[30] which resulted in (89  9) % of the receptor being capable of binding the ligand. The standard method to investigate membrane protein dynamics is static 15N cross-polarization (CP) NMR spectroscopy.[20] From the lineshape of the NMR spectra, conclusions about the motional averaging of the 15N chemical-shift tensor of the backbone and side chain amides can be drawn. Figure 1 shows characteristic static 15N NMR spectra of the U–15N labeled Y2 receptor at 30 and 37 8C. At 30 8C, only a small shoulder at high field indicates mobile lysine (Lys) NH3 + side chains (Figure 1A). At 378, however, some significant alterations of the spectra occur. Whereas most amides show a characteristic axially symmetric rotational diffusion of the entire receptor in the membrane with the main principle values for the 15N shielding tensor of s j j  227 ppm and s ?  82 ppm, several isotropic signals are detected that are indicative of residues undergoing large amplitude fluctuations in their backbone and/or side chain (Figure 1B). These mobile residues can be identified if the CP contact time is systematically altered. At short CP contact time, only the rigid sites will be cross-polarized; the mobile sites require longer contact times for efficient polarization transfer. At a short contact time of 70 ms (Figure 1C), no mobile residues are identified, except for a small signal for the Lys NH3 + . However, the situation changes completely when longer contact times are used. At 2 ms contact time, in addition to a prominent Lys NH3 + peak at d  35 ppm, the characteristic arginine (Arg) Ne and Arg Nh signals at d  89 and 75 ppm are seen in addition to two isototropic signals in the amide region at d  123 and 119 ppm (Figure 1B). These mobile residues are detected with much higher intensity when the CP contact time is increased to 8 ms (Figure 1D), which indicates the dynamic bias of the polarization transfer by CP. The two isotropic signals account for about 30 % of the total spectral intensity of the 15N powder spectrum (Figure S1 in the Supporting Information), which suggests that about one-quarter of the amides is nearly isotropically mobile. Such significant molecular mobility of individual amino acids in the Y2 receptor should also improve the spectral resolution

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Full Paper in 15N magic-angle spinning (MAS) NMR spectra. Characteristic 15 N MAS NMR spectra of the reconstituted Y2 receptor are shown in Figure S2 in the Supporting Information. In the CPMAS NMR spectra, the NH residues show a relatively broad and featureless line with a shoulder at high field that is indicative of the NH2 side chains of glutamine (Gln) and asparagine (Asn). The only narrow lines are assigned to Lys NH3 + and Arg Ne and Nh signals. However, it should be emphasized that CP spectra are biased towards residues in rigid sites that feature larger 1H,15N dipolar couplings. Therefore, we also recorded 15N MAS NMR spectra that were polarized by using the insensitive nuclei enhanced by polarization transfer (INEPT) pulse sequence (Figure S2B in the Supporting Information). Clearly narrower NMR spectra are detected that show separated NH and NH2 sites. Two-dimensional 1H,15N heteronuclear correlation (HetCor) experiments using INEPT polarization transfer show a relatively well-resolved NH2 region as well as a glycine (Gly) region that is thoroughly separated from the rest of the amide residues (Figure S3A in the Supporting Information). Nevertheless, the overall resolution of this spectrum is poor. In contrast, 1 15 H, N HetCor spectra excited by a CP are very broad, show poor resolution, and the dispersion of the 1H signals in the indirect dimension covers approximately 30 kHz (Figure S3B in the Supporting Information). Next, we investigated the molecular dynamics of the membrane-reconstituted Y2 receptor by 13C MAS NMR. Figure 2 shows characteristic 13C CP-MAS (Figure 2A) and directly excited 13C MAS NMR spectra (Figure 2B). The 13C NMR spectra show better signal dispersion and resolution than the 15N spectra, and the signals of the aliphatic CH, CH2, and CH3 carbons can be well separated. Subtle differences between 13C CP-MAS

Figure 2. 13C MAS NMR spectra of the uniformly 13C-labeled Y2 receptor in [D54]DMPC membranes using different polarization pulse sequences. A) CPMAS spectrum using a CP contact time of 0.7 ms, B) directly excited 13C MAS NMR spectrum, and C) 13C INEPT MAS NMR spectrum. All spectra were recorded at 37 8C at a MAS frequency of 7 kHz using Spinal64 decoupling with a radio frequency amplitude of approximately 65 kHz. Chem. Eur. J. 2014, 20, 4986 – 4992

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and directly excited MAS spectra suggest that parts of the Y2 receptor undergo large amplitude motions in the membrane. In particular, the CO region shows more sharp lines in the directly excited spectrum than the CP-MAS spectra. This is also observed for the CH2 region. To further quantify these interesting effects, we investigated the molecular dynamics of the Y2 receptor by measuring the motionally averaged 1H,13C dipolar couplings using the separated local-field experiment DIPSHIFT.[31] The ratio of motionally averaged and full dipolar coupling defines an order parameter for the HC bond vector that ranges from 0 for isotropic mobility to 1 for rigidity. Order parameters could be determined for the backbone CH region, the well-resolved Gly residues, and the CH2 as well as the CH3 side chains; these are displayed in Figure 3A. At a short CP contact time of 20 ms, order parameters are relatively high for all backbone and side-chain signals (0.88 for CH, 0.66 for CH2, and 0.46 for CH3). If the CP contact time is increased, the more mobile sites will be sufficiently cross-polarized as well and contribute to the spectral intensity, thereby resulting in a decrease in the order parameters. At a long contact time of 2 ms, order parameters are significantly lower for all investigated sites. To excite all residues homogeneously, directly excited DIPSHIFT experiments were carried out. These directly excited DIPSHIFT spectra provide an unbiased view of the receptor dynamics. Under these conditions, the lowest-order parameters of 0.55 for CH, 0.58 for Gly, 0.33 for CH2, and 0.17 for CH3 were measured at 23 8C (see Table S1 in the Supporting Information for order-parameter measurements at all temperatures investigated). Such low-order parameters are indicative of individual backbone and side-chain sites undergoing relatively large amplitude motions. Temperaturedependent order-parameter measurements show that the segmental mobility of the Y2 receptor follows a simple Arrhenius behavior as demonstrated in a semilogarithmic plot of the order parameter against the inverse temperature. From the slope of these plots, activation energies between 2.7 and 4.3 kJ mol1 could be determined. The phase transition of the DMPC membrane at 23 8C does not induce any significant deviation from the Arrhenius behavior. It was confirmed by differential scanning calorimetry (DSC) measurements that the phase transition of DMPC in the absence and in the presence of the Y2 receptor occurred at approximately the same temperature (Figure S4 in the Supporting Information). As these order-parameter studies show, the Y2 receptor in membranes is highly mobile and undergoes rapid rotational diffusion in the membrane as well as large amplitude segmental motions. This should enable detection of the mobile residues through 1H,13C INEPT polarization transfer using scalar couplings. The resulting 13C INEPT NMR spectrum is shown in Figure 3C; it displays quite a significant number of narrow lines in the well-resolved NMR spectrum of the Y2 receptor. In the 2D 1H,13C INEPT spectrum shown in Figure S5 of the Supporting Information, many individually resolved peaks are detected, including backbone and side-chain chemical shifts. The NMR spectroscopic signals from the lipid can be satisfactorily assigned, as only the headgroup and glycerol backbone sites in chain-perdeuterated [D54]DMPC contribute to the spectrum.

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Figure 4. Static 2H NMR spectrum of uniformly 2H-labeled Y2 receptor in DMPC membrane at a temperature of A) 30 8C and B) 23 8C. The simulation of the spectral lineshape at 30 8C from two Pake doublets with order parameters of 0.97 and 0.3, respectively, is shown in gray. Spectrum (B) was scaled to highlight the anisotropic features of the spectrum to assess the relative contribution from the isotropic peak; the full NMR spectrum is shown in the inset. Spectra were recorded in protonated DMPC membranes. A sample with the same amount of DMPC but no receptor present did not provide a natural-abundance 2H NMR spectrum within the same experimental time.

cative of fully rigid segments of the Y2 receptor. The quadrupolar splitting of this spectral component remained constant at all investigated temperatures.

Discussion 13

Figure 3. C NMR spectroscopic order parameters for backbone and sidechain carbon atoms of the uniformly 13C-labeled Y2 receptor in DMPC membranes at A) varying CP contact times and B) temperatures. CH dipolar coupling constants were measured using DIPSHIFT experiments and averaged over several signals in the same 13C chemical-shift region, except for the glycine Ca signal. In panel (A), order parameters were determined at a temperature of 23 8C. For panel (B), the CP contact time was 0.7 ms. Lines in panel (B) represent linear regressions.

The 1H linewidth of the receptor signals in these spectra is 130–180 Hz, which is comparable to highly mobile membraneassociated proteins.[32] Finally, static 2H NMR spectra of the fully deuterated Y2 receptor were recorded (Figure 4). At 30 8C, the NMR spectrum can be explained by a superposition of two Pake doublets with a quadrupolar splitting of 120 kHz (S = 0.97) and 40 kHz (S = 0.32). At physiological temperature of 37 8C, quite significant changes of the spectral line shape are detected. Overall, a relatively narrow 2H NMR spectrum was detected that features a maximal width of approximately 40 kHz, which is indicative of a maximum order parameter of S  0.32. No individual quadrupolar splittings are resolved and the intensity increase towards the center of the spectrum merges into a large isotropic peak. The lineshape is reminiscent of the 2H NMR spectra of fast two-site exchange.[33] In addition to the narrow component of the NMR spectrum, a Pake spectrum with a large quadrupolar splitting of 120 kHz is also detected, which is indiChem. Eur. J. 2014, 20, 4986 – 4992

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GPCRs are highly dynamic molecules, and the difficulties that have been encountered in crystallizing these membrane proteins over the last decades underline this fact. Up to now, all GPCR crystals have only been achieved by immobilizing the molecule by anti- or nanobody binding, by replacing or removing loops, or by thermostabilization through mutations.[1] Nevertheless, the crystallographic B factors are still quite high for GPCRs and range from 30–120 2 for the transmembrane helices to 100–280 2 for the loops and termini. Here, we have studied the molecular dynamics of the full-length human neuropeptide Y receptor type 2 reconstituted into DMPC membranes by means of solid-state NMR spectroscopy. We used a set of experiments sensitive to molecular motions on different timescales with the upper limits in the microsecond correlation time window. Static 15N CP NMR spectra are influenced by motions with correlation times faster than approximately 100 ms.[20] The 15N NMR spectra of the Y2 receptor in membranes were dominated by axially symmetric motions of the whole molecule. According to the Saffman–Delbrck theory,[34] a-helical proteins of the size of GPCRs should undergo such rotational diffusion with correlation times of approximately 0.4 ms. In addition to the rotation of the entire Y2 receptor, segmental motions with large amplitudes have been observed that give rise to isotropic signals in static 15N CP and 2H NMR spectra both in the side

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Full Paper chains and in the backbone. As polarization transfer by CP is biased towards rigid residues that feature large dipolar couplings, longer CP contact times had to be employed to detect the more mobile sites. Under these experimental conditions, very prominent narrow signals dominate the 15N NMR spectra, which most likely stem from highly mobile loop and tail structures of the membrane-embedded receptor.[4, 35] More quantitative information on dynamics is available from 1 H–13C dipolar coupling measurements. H–C dipolar couplings have a maximum strength of 22.8 kHz and are therefore sensitive to motions with correlation times of up to approximately 40 ms. By increasing the CP contact time, the mobile sites of the receptor that feature small dipolar couplings are cross-polarized as well so that lower-order parameters are observed (Figure 3, Table S1 in the Supporting Information). Directly excited DIPSHIFT measurements reveal order parameters of 0.55, 0.33, and 0.17 for the CH, CH2, and CH3 segments of the receptor, respectively. Such values are significantly lower than those found for colicin Ia, for example. This membrane-bound protein featured order parameters of 0.88–0.93 for the backbone and 0.6–0.75 in the side chain, which was, however, determined at a short CP contact time of 0.7 ms.[36] The seven transmembrane helical protein Anabaena sensory rhodopsin also features order parameters between 0.9 and 1.0 for the majority of the residues.[37] Relatively low-order parameters of approximately 0.8 have also been found for most sites in crystalline GB1[38] or ubiquitin;[39] in the latter, two single residues showed order parameters as low as 0.45 and 0.56, respectively. Whereas crystalline ubiquitin was pronounced “highly dynamic on the microsecond to picosecond time scale”,[39] we note that the overall mobility of the Y2 GPCR in DMPC membranes is significantly higher. Although the current resolution of our solidstate NMR spectra does not allow for site-specific assignments, some interesting conclusions about the overall mobility of the protein in the membrane can be drawn. Assuming a wobbling in a cone model to convert order parameters into motional amplitudes,[17] the Y2 receptor undergoes fluctuations with amplitudes as high as approximately 508 in the backbone and approximately 50–608 in the side chain, not considering the methyl rotation of the CH3 groups. Most of these dynamics are segmental and underline the large intrinsic mobility of the receptor in the membrane. In addition to fluctuations of the individual residues, motions of the transmembrane a-helices relative to each other can contribute to the dynamics in this correlation time window. The segmental molecular mobility of the Y2 receptor is intrinsic and does not seem to receive activation from the membrane lipids. Temperature-dependent order-parameter measurements show an Arrhenius behavior irrespective of the membrane phase state. This suggests that the Y2 receptor is also relatively mobile in the gel state of the membrane. Whereas GPCR function is highly dependent on the properties of the surrounding lipid matrix,[40, 41] the basal dynamics of the molecule do not appear to depend on the membrane phase state. The activation energy for these segmental motions is only on the order of kBT, thus reflecting the structural flexibility of the membrane-embedded GPCR molecules. Chem. Eur. J. 2014, 20, 4986 – 4992

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H NMR spectra probe the largest NMR spectroscopic interaction and are therefore sensitive to motions with correlation times of up to approximately 6 ms. The rotational diffusion of the receptor is still contained in this time window. At 30 8C, the 2H NMR spectrum can simply be explained by rigid CH and CH2 as well as rotating CH3 groups. This is in agreement with the 13C order-parameter measurements at this temperature (Table S1 in the Supporting Information). However, at 37 8C, most of the molecular segments of the receptor undergo motions with order parameters of  0.3. The 2H NMR spectra of the Y2 receptor at 37 8C also showed a contribution of very rigid segments that was indicative of a rigid internal core of the receptor at physiological temperature. It is likely that this spectral component is represented by the transmembrane ahelices. Owing to the excitation profile of the 4 ms 2H excitation pulses, the powder spectra cannot fully be quantified. Our data can provide a model for the dynamic behavior of the Y2 receptor in liquid-crystalline membranes. If we roughly assume that the 194 amino acids of the loops and tails of the receptor show an average order parameter of 0.3 and the remaining 187 amino acids that form the a-helices exhibit average order parameters of 0.9 in the backbone, as suggested by the 2H NMR spectroscopic measurements, the average order parameter of the receptor protein backbone would equal approximately 0.6, which agrees very well with average order parameter of 0.55 measured for the backbone in the 1H,13C dipolar coupling experiments (see Figure 3). Thus, a possible interpretation of our data is that the loops and tails of the receptor are highly mobile, whereas the transmembrane a-helices show a more rigid local structure that undergoes rotational diffusion in the membrane. Such a model also seems to apply for sensory rhodopsin II from Natronomomonas pharaonis.[42] Considering such a high mobility of the Y2 receptor, one would expect that the 1H,X correlation experiments would provide relatively well-resolved NMR spectra. For 15N detection, this was not observed. CP-based HetCor experiments showed no 1H resolution whatsoever, and the 1H,15N INEPT HetCor NMR spectra also featured broad lineshapes of low resolution, which suggests that short T2 relaxation times dominate the spectral shape. As axially symmetric rotational diffusion represents a dominant mechanism of the receptor dynamics, this motion does not contribute much to the relaxation mechanisms as many amides are localized in the transmembrane a-helices with HN bond vectors relatively parallel to the symmetry axis of the rotation. Consequently, 1H,15N HSQC spectra of GPCRs in solution show relatively large linewidths.[43, 44] In contrast, 1H,13C correlation experiments show a somewhat better resolution and display quite a number of well-resolved cross-peaks, which most likely come from the highly mobile termini of the receptor.[4] Apparently, the axially symmetric rotations along with the segmental mobility of the receptor provide a significant reduction in the dipolar couplings along with a sufficient increase in relaxation times. Thus, the high-resolution MAS NMR spectroscopic techniques might be advantageous for the investigation of GPCRs in membranes. EPR work on rhodopsin has contributed a wealth of data on dynamics for this GPCR. On the basis of the EPR spectra, a pa-

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Full Paper rameter of scaled mobility Ms can be defined that roughly lies between 0 and 1.[10] It has been shown that Ms and the solution NMR spectroscopic order parameter of the NH bond S2 are related according to Ms  1S2.[10] From our backbone order-parameter measurements, we determined an average order parameter of 0.55, which would then correspond to a scaled mobility parameter of Ms  0.7, which agrees with what has been found in EPR measurements for the loop and tail structures of rhodopsin.[10–14] However, it should be remembered that the NMR spectroscopic order-parameter measurement samples motions on a longer timescale (105 s) than EPR (108 s), which explains why the EPR showed such high mobility exclusively in the loops and tails. Previous work on the molecular dynamics of proteorhodopsin, a bacterial heptahelical membrane protein with similar topology to a GPCR, has shown that hydration plays the decisive role in enhancing the molecular fluctuations of the tails and loops.[45] Residues that undergo large amplitude fluctuations on timescales < 105 s were termed J residues, whereas segments with smaller amplitude motions were referred to as D residues. Accordingly, these segments could be detected by either INEPT or cross-polarization transfer schemes. J residues were found in the termini, turns, as well as at the ends of the transmembrane a-helices. We can adapt this nomenclature to the Y2 GPCR as well, which features a number of highly mobile J residues along with quite mobile D residues. The latter may also be localized in the transmembrane segments of the molecule, which feature 7 Gly and 7 Pro residues overall; this should also impart some flexibility to these secondary structure elements.

Conclusion The human neuropeptide Y receptor type 2 reconstituted in DMPC was found to undergo fast rotational diffusion in the membrane and fluctuations with varying amplitudes including large amplitude motions in the backbone and the side chains. These equilibrium dynamics provide the molecule with structural plasticity that is required for conformational changes upon receptor activation. On a timescale up to microseconds, the motional amplitudes of the receptor backbone and side chain reach values between approximately 508 and approximately 50–608, respectively, on average. The study presented here provides a first benchmark for dynamic parameters of a non-rhodopsin GPCR in a nativelike environment.

Experimental Section Expression of the Y2 receptor A cysteine-deficient variant of the human Y2 receptor, in which all cysteines except the two in the conserved disulfide bridge were replaced by alanine (Ala) or serine (Ser),[46] was expressed in E. coli as inclusion bodies in a defined mineral salt medium by using a fedbatch-fermentation process as described before.[47–49] Uniform 15Nlabeling of the receptor was achieved by using 15NH3Cl and 15 NH3(SO4)2 (Cambridge Isotope Lab., USA) as the sole nitrogen source in the fermentation medium. For the expression of the UChem. Eur. J. 2014, 20, 4986 – 4992

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C-/15N-labeled receptor, U-13C6 glucose (Cambridge Isotope Lab., USA) was applied to the medium in the feed phase, starting 30 min prior to induction (30 g L1 in total). Expression time was shortened to 3 h to save labeled glucose. For the expression of the U-2H-/15N-labeled receptor, the transformed cells were adapted to 80 % D2O in several steps starting from 50 %. Because of reduced cell growth rates in D2O, the expression time after induction was extended to 6 h. Expression yields reached 22, 18, and 13 mg L1 medium for the U-15N, U-13C-/15N-, and U-2H-/15N-labeled receptors, respectively. Inclusion body preparation, solubilization of the receptor molecules in sodium dodecyl sulfate (SDS), and receptor purification were performed as described before.[50]

NMR spectroscopic sample preparation Purified Y2 receptor, solubilized in 15 mm SDS and 50 mm sodium phosphate at pH 8, was diluted to 0.5 mg mL1 and dialyzed against a buffer that contained 2 mm SDS, 50 mm sodium phosphate, 1 mm ethylenediaminetetraacetic acid (EDTA), 0.1 mm reduced glutathione (GSH), and 0.05 mm oxidized glutathione (GSSG) at pH 8.5 for 48 h for the formation of the functional disulfide bridge.[51] For reconstitution, unilamellar vesicles with a diameter of 100 nm were prepared by extrusion[52] in 50 mm sodium phosphate buffer at pH 8 and a [D54]DMPC concentration of 10 mg mL1. These vesicles were solubilized with a fourfold molar excess amount of DHPC-c7 (Avanti Polar Lipids, USA).[53] Then the Y2 receptor was incubated with the detergent/lipid mix at molar ratio of 1:180:720 receptor/DMPC/DHPC-c7, followed by three cycles of fast temperature changes between 40 and 0 8C (25 min each step).[54] After the temperature cycles, BioBeadsSM2 (Bio-Rad Lab., Germany; 75 mg mL1) were added twice to the solution to remove the detergent. The concentration of detergents was checked by 1H MAS NMR spectroscopy and found to be < 5 % DHPC-c7 and < 1 % SDS of the total lipid. The resulting cloudy receptor/DMPC dispersion was ultra-centrifuged at 86 000  g, overnight. The final NMR spectroscopic sample contained approximately 6 mg Y2 receptor at a receptor-to-lipid molar ratio of approximately 1:175 and a water content of approximately 70 wt %.

NMR spectroscopic experiments Standard static 15N CP NMR spectra with Hahn echo detection were acquired using a Bruker Avance I 750 MHz NMR spectrometer and a double-channel probe with a 5 mm coil. The CP contact time varied between 70 ms and 8 ms; typical 908 pulses were 4 ms for 1H and 6 ms for 15N. During signal detection, heteronuclear twopulse phase modulated (TPPM) decoupling[55] was applied with a radio-frequency field strength of approximately 62 kHz. All 13C and 15N MAS NMR spectroscopic experiments were carried out using a Bruker Avance III 600 NMR spectrometer with a double-resonance MAS probe equipped with a 4 mm spinning module. Typical 908 pulse lengths were 4 ms for 1H and 13C, and 5 ms for 15N. The 1 H radio-frequency field strength during heteronuclear decoupling using Spinal64[56] was approximately 65 kHz. The 13C/15N chemical shifts were referenced using external standards.[57] Standard CPMAS, HetCor, and INEPT experiments were carried out at a MAS frequency of 7 kHz. In the 1H dimensions, between 140 and 400 increments were acquired. The 13C,1H dipolar couplings strength was measured in DIPSHIFT experiments.[31] Frequency-switched Lee– Goldburg (FSLG) sequence[58] was used for 1H,1H homonuclear decoupling. Either direct excitation or CP with varying contact times between 20 ms and 2 ms was used. The signal was acquired only over one rotor period in the indirect dimension, and the dipolar

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Full Paper dephased signal was extracted for each resolved peak and fitted to yield the coupling strength and determine order parameters. 2 H NMR spectra of the U-2H-/15N-labeled receptor were acquired using a Bruker Avance I 750 MHz NMR spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) operating at a resonance frequency of 115.1 MHz for 2H NMR spectra. A single-channel solids probe equipped with a 5 mm solenoid coil was used. The 2H NMR spectra were accumulated with a spectral width of  250 kHz using the quadrupolar echo sequence. The typical length of a 908 pulse was 4.0 ms, and a relaxation delay of 1 s was applied.

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Received: December 17, 2013 Revised: January 20, 2014 Published online on March 13, 2014

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