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• W2554301698 abstract "Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031–4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1–TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187–3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior. Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031–4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1–TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187–3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior. G-protein coupled receptors (GPCRs) 3The abbreviations used are: GPCR, G-protein coupled receptor; TM, transmembrane; ER, endoplasmic reticulum; RP-HPLC, reverse phase-HPLC; LPPG, 1-palmitoyl-2-hydroxy-sn-glycero-3-(phospho-rac-(1-glycerol)); DPC, dodecyl-phosphocholine; SEC-MALS, size exclusion chromatographymultiangle light scattering; RDC, residual dipolar coupling; PRE, paramagnetic relaxation enhancement; MTSL, S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate; Gd-(DTPA-BMA), gadolinium diethylenetriamine-pentaacetic acid bis-methyl-amine; MSP, membrane scaffold protein; PG, phosphatidylglycerol; BR, bacteriorhodopsin; OST, oligosaccharyl transferase; Endo H, endoglycosidase H; DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; HFIP, hexafluoroisopropanol; RM, rough microsomes; HSQC, heteronuclear single quantum coherence; OST, oligosaccharyl transferase. are a large family of integral membrane proteins that transmit signals into cells upon activation by a set of highly chemically heterogeneous inducers (1.Lefkowitz R.J. A brief history of G-protein coupled receptors (Nobel Lecture).Angew. Chem. Int. Ed. Engl. 2013; 52: 6366-6378Crossref PubMed Scopus (187) Google Scholar). GPCRs form a seven-transmembrane (TM) helical bundle wherein the individual helices are connected by three extracellular and intracellular loops. The helix bundle is attached to an extracellular N-terminal domain of highly variable size and structure, and an intracellular and mostly flexible C terminus that often contains an eighth helix (2.Kobilka B. The structural basis of G-protein-coupled receptor signaling (Nobel Lecture).Angew. Chem. Int. Ed. Engl. 2013; 52: 6380-6388Crossref PubMed Scopus (133) Google Scholar, 3.Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Crystal structure of rhodopsin: a G protein-coupled receptor.Science. 2000; 289: 739-745Crossref PubMed Scopus (5023) Google Scholar4.Venkatakrishnan A.J. Deupi X. Lebon G. Tate C.G. Schertler G.F. Babu M.M. Molecular signatures of G-protein-coupled receptors.Nature. 2013; 494: 185-194Crossref PubMed Scopus (1088) Google Scholar). Binding of an extracellular agonist stabilizes the conformation of the receptor that activates the now more accessible heterotrimeric G-protein (5.Rasmussen S.G. Choi H.J. Rosenbaum D.M. Kobilka T.S. Thian F.S. Edwards P.C. Burghammer M. Ratnala V.R. Sanishvili R. Fischetti R.F. Schertler G.F. Weis W.I. Kobilka B.K. Crystal structure of the human β2 adrenergic G-protein-coupled receptor.Nature. 2007; 450: 383-387Crossref PubMed Scopus (1665) Google Scholar). The first high-resolution structure of a GPCR, that of bovine rhodopsin, was published in 2000 (3.Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Crystal structure of rhodopsin: a G protein-coupled receptor.Science. 2000; 289: 739-745Crossref PubMed Scopus (5023) Google Scholar), followed in 2007 by the first X-ray structure of a recombinantly produced GPCR (6.Cherezov V. Rosenbaum D.M. Hanson M.A. Rasmussen S.G. Thian F.S. Kobilka T.S. Choi H.J. Kuhn P. Weis W.I. Kobilka B.K. Stevens R.C. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor.Science. 2007; 318: 1258-1265Crossref PubMed Scopus (2762) Google Scholar). Subsequently, many structures of GPCRs in ground and activated states have been published (2.Kobilka B. The structural basis of G-protein-coupled receptor signaling (Nobel Lecture).Angew. Chem. Int. Ed. Engl. 2013; 52: 6380-6388Crossref PubMed Scopus (133) Google Scholar, 4.Venkatakrishnan A.J. Deupi X. Lebon G. Tate C.G. Schertler G.F. Babu M.M. Molecular signatures of G-protein-coupled receptors.Nature. 2013; 494: 185-194Crossref PubMed Scopus (1088) Google Scholar). Importantly, coordinates of an agonist-bound GPCR coupled to a G-protein have been released (7.Rasmussen S.G. DeVree B.T. Zou Y. Kruse A.C. Chung K.Y. Kobilka T.S. Thian F.S. Chae P.S. Pardon E. Calinski D. Mathiesen J.M. Shah S.T. Lyons J.A. Caffrey M. Gellman S.H. et al.Crystal structure of the β2 adrenergic receptor-Gs protein complex.Nature. 2011; 477: 549-555Crossref PubMed Scopus (2267) Google Scholar), and the structure of a GPCR-arrestin complex was solved (8.Kang Y. Zhou X.E. Gao X. He Y. Liu W. Ishchenko A. Barty A. White T.A. Yefanov O. Han G.W. Xu Q. de Waal P.W. Ke J. Tan M.H. Zhang C. et al.Crystal structure of rhodopsin bound to arrestin by fs X-ray laser.Nature. 2015; 523: 561-567Crossref PubMed Scopus (578) Google Scholar). Although our knowledge of the structure of GPCRs in various states and their mode of activation and desensitization is rapidly increasing, detailed information on their folding pathways is still lacking. The popular refined two-stage model from Popot and Engelman (9.Engelman D.M. Chen Y. Chin C.N. Curran A.R. Dixon A.M. Dupuy A.D. Lee A.S. Lehnert U. Matthews E.E. Reshetnyak Y.K. Senes A. Popot J.L. Membrane protein folding: beyond the two stage model.FEBS Lett. 2003; 555: 122-125Crossref PubMed Scopus (255) Google Scholar, 10.Popot J.L. Engelman D.M. Membrane protein folding and oligomerization: the two-stage model.Biochemistry. 1990; 29: 4031-4037Crossref PubMed Scopus (821) Google Scholar11.Popot J.L. Engelman D.M. Helical membrane protein folding, stability, and evolution.Annu. Rev. Biochem. 2000; 69: 881-922Crossref PubMed Scopus (538) Google Scholar) postulates that secondary structures form when the peptide chain partitions into the membrane-water interface. However, proteins destined for membrane insertion are generally subjected to the concerted action of translating ribosomes in the cytoplasm and translocon complexes located in the endoplasmic reticulum (ER) of eukaryotes or in the plasma membrane of bacteria (12.White S.H. von Heijne G. How translocons select transmembrane helices.Annu. Rev. Biophys. 2008; 37: 23-42Crossref PubMed Scopus (162) Google Scholar). To traffic proteins to membranes in most cells the signal-recognition particle targets the nascent chains emerging from the ribosome tunnel to the translocon complex (13.Martínez-Gil L. Saurí A. Marti-Renom M.A. Mingarro I. Membrane protein integration into the endoplasmic reticulum.FEBS J. 2011; 278: 3846-3858Crossref PubMed Scopus (28) Google Scholar). After folding within the ribosome-translocon complex (14.Bhushan S. Gartmann M. Halic M. Armache J.P. Jarasch A. Mielke T. Berninghausen O. Wilson D.N. Beckmann R. α-Helical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel.Nat. Struct. Mol. Biol. 2010; 17: 313-317Crossref PubMed Scopus (156) Google Scholar, 15.Mingarro I. Nilsson I. Whitley P. von Heijne G. Different conformations of nascent polypeptides during translocation across the ER membrane.BMC Cell Biol. 2000; 1: 3Crossref PubMed Scopus (71) Google Scholar16.Woolhead C.A. McCormick P.J. Johnson A.E. Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins.Cell. 2004; 116: 725-736Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar), individual helices will then insert into and diffuse laterally in the membrane until native contacts are formed and the bundle fully assembles. In this co-translational insertion/folding process, segments of sufficient hydrophobicity are laterally gated from the translocon into the membrane interior. A remarkable agreement has been observed between the purely biophysical Wimley-White scale (17.Wimley W.C. White S.H. Experimentally determined hydrophobicity scale for proteins at membrane interfaces.Nature Struct. Biol. 1996; 3: 842-848Crossref PubMed Scopus (1396) Google Scholar) and the biological translocon scale from the von Heijne group (18.Hessa T. Meindl-Beinker N.M. Bernsel A. Kim H. Sato Y. Lerch-Bader M. Nilsson I. White S.H. von Heijne G. Molecular code for transmembrane-helix recognition by the Sec61 translocon.Nature. 2007; 450: 1026-1030Crossref PubMed Scopus (535) Google Scholar). This agreement suggests that formation of the TM helices and their insertion into the membrane are largely governed by thermodynamic factors that are related to the amino acid sequence of the GPCRs (12.White S.H. von Heijne G. How translocons select transmembrane helices.Annu. Rev. Biophys. 2008; 37: 23-42Crossref PubMed Scopus (162) Google Scholar, 18.Hessa T. Meindl-Beinker N.M. Bernsel A. Kim H. Sato Y. Lerch-Bader M. Nilsson I. White S.H. von Heijne G. Molecular code for transmembrane-helix recognition by the Sec61 translocon.Nature. 2007; 450: 1026-1030Crossref PubMed Scopus (535) Google Scholar, 19.Baeza-Delgado C. von Heijne G. Marti-Renom M.A. Mingarro I. Biological insertion of computationally designed short transmembrane segments.Sci. Rep. 2016; 6: 23397Crossref PubMed Scopus (15) Google Scholar20.White S.H. Translocons, thermodynamics, and the folding of membrane proteins.FEBS Lett. 2003; 555: 116-121Crossref PubMed Scopus (49) Google Scholar). Folding of polytopic membrane proteins is the result of a series of events that include helix insertion into the hydrophobic core and sequestering of loop sequences into cytosolic or extracellular space (11.Popot J.L. Engelman D.M. Helical membrane protein folding, stability, and evolution.Annu. Rev. Biochem. 2000; 69: 881-922Crossref PubMed Scopus (538) Google Scholar, 21.Whitley P. Mingarro I. Stitching proteins into membranes, not sew simple.Biol. Chem. 2014; 395: 1417-1424Crossref PubMed Scopus (8) Google Scholar). The timing of the chain insertion and the localization of TM helices would be expected to be a consequence of the amino acid sequence and the interaction of the growing polypeptide chain with the membrane. Recently, however, the first evidence was obtained that helices might change their location during synthesis of later portions of the polypeptide chain (22.Bowie J.U. Structural biology: membrane protein twists and turns.Science. 2013; 339: 398-399Crossref PubMed Scopus (27) Google Scholar, 23.Lu Y. Turnbull I.R. Bragin A. Carveth K. Verkman A.S. Skach W.R. Reorientation of aquaporin-1 topology during maturation in the endoplasmic reticulum.Mol. Biol. Cell. 2000; 11: 2973-2985Crossref PubMed Scopus (104) Google Scholar24.Virkki M.T. Agrawal N. Edsbäcker E. Cristobal S. Elofsson A. Kauko A. Folding of Aquaporin 1: multiple evidence that helix 3 can shift out of the membrane core.Protein Sci. 2014; 23: 981-992Crossref PubMed Scopus (16) Google Scholar) emphasizing the aspect of context for proper folding. An apparent conceptual problem with the sequential, hydrophobicity-based, folding model is that TM helices of some membrane proteins are only marginally hydrophobic. Helix-bundle membrane proteins display a large number of inter-helical contacts that are often formed between polar or even charged residues (25.Bañó-Polo M. Martínez-Gil L. Wallner B. Nieva J.L. Elofsson A. Mingarro I. Charge pair interactions in transmembrane helices and turn propensity of the connecting sequence promote helical hairpin insertion.J. Mol. Biol. 2013; 425: 830-840Crossref PubMed Scopus (25) Google Scholar26.Chamberlain A.K. Faham S. Yohannan S. Bowie J.U. Construction of helix-bundle membrane proteins.Adv. Protein Chem. 2003; 63: 19-46Crossref PubMed Scopus (44) Google Scholar, 27.Gratkowski H. Lear J.D. DeGrado W.F. Polar side chains drive the association of model transmembrane peptides.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 880-885Crossref PubMed Scopus (289) Google Scholar28.Senes A. Engel D.E. DeGrado W.F. Folding of helical membrane proteins: the role of polar, GxxxG-like and proline motifs.Curr. Opin. Struct. Biol. 2004; 14: 465-479Crossref PubMed Scopus (356) Google Scholar). We noticed that some TM segments of GPCRs that are under study in our lab do not display favorable energies for full membrane insertion (29.Marino J. Geertsma E.R. Zerbe O. Topogenesis of heterologously expressed fragments of the human Y4 GPCR.Biochim. Biophys. Acta. 2012; 1818: 3055-3063Crossref PubMed Scopus (6) Google Scholar, 30.Shao X. Zou C. Naider F. Zerbe O. Comparison of fragments comprising the first two helices of the human Y4 and the yeast Ste2p G-protein-coupled receptors.Biophys. J. 2012; 103: 817-826Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), an observation not unlike that reported for individual TMs of bacteriorhodopsin (31.Kahn T.W. Engelman D.M. Bacteriorhodopsin can be refolded from two independently stable transmembrane helices and the complementary five-helix fragment.Biochemistry. 1992; 31: 6144-6151Crossref PubMed Scopus (144) Google Scholar). It is therefore highly questionable whether in the absence of other interacting helices these “hydrophilic” TM segments would still fully insert into the membrane. To answer whether insertion occurs, topology-mapping methods that use terminally fused reporter moieties have been developed by the von Heijne group (32.Drew D. Sjöstrand D. Nilsson J. Urbig T. Chin C.N. de Gier J.W. von Heijne G. Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 2690-2695Crossref PubMed Scopus (170) Google Scholar). Although they very successfully allow the rapid attainment of a quantitative picture of the location of the TM termini, they unfortunately do not provide any detailed structural information on the TM segments or on protein-membrane interactions. To address some of these issues we have developed a systematic approach to investigate conformational preferences of N-terminal fragments of the Ste2p receptor, a yeast GPCR, using solution NMR methods. Considering that proteins are synthesized starting with the N terminus of the polypeptide chain, TM1 is expected to be the first segment that is inserted into the hydrophobic core, followed by TM2 and so on (21.Whitley P. Mingarro I. Stitching proteins into membranes, not sew simple.Biol. Chem. 2014; 395: 1417-1424Crossref PubMed Scopus (8) Google Scholar). We therefore report on studies of polypeptides corresponding to the overlapping fragments TM1, TM1–TM2 (TM12), and TM1–TM2–TM3 (TM123) (supplemental Fig. S1). TM1, TM2, and TM7 form a distinct subcore in the fully assembled helix bundle, and, based on analysis of published GPCR structures, often more contacts exist between TM7 and TM1 and TM2 than between TM3 and TM2 (4.Venkatakrishnan A.J. Deupi X. Lebon G. Tate C.G. Schertler G.F. Babu M.M. Molecular signatures of G-protein-coupled receptors.Nature. 2013; 494: 185-194Crossref PubMed Scopus (1088) Google Scholar). Therefore, we have also examined a chimeric three TM helix construct, TM1–TM2–TM7 (TM127). In this construct the N-terminal end of TM7 is linked to TM2 using portions of IL1 (intracellular loop 1) and EL3 (extracellular loop 3, supplemental Fig. S1). NMR is being used to determine conformational preferences of these Ste2p fragments and their overall topology in detergent micelles. As the size of these polypeptides has increased from 60–80 (TM12) to 160–180 (3-TM constructs) residues, the NMR assignments became more challenging. We, therefore, have probed whether chemical shift assignments, which are more easily obtained for smaller fragments, can be transferred to these larger fragments. To ensure that no significant artifacts are introduced by conducting NMR studies in micelles, which do not represent true bilayers, we have also investigated TM127 incorporated into nanodiscs with different lipid compositions. Finally, we have monitored insertion of all these constructs into ER-derived membranes using an established insertion-glycosylation assay to obtain data on their integration into true biological membranes. Our data indicate that TM insertion is indeed context-dependent. The Ste2p fragments TM1 (Gly31-Thr78), TM12 (Gly31-Thr110), TM123 (Gly31-Arg161), and TM127 (Gly31-Thr114, Thr274-Leu340) were expressed as C-terminal fusions to the hydrophobic TrpΔLE sequence (33.Miozzari G.F. Yanofsky C. Translation of the leader region of the Escherichia coli tryptophan operon.J. Bacteriol. 1978; 133: 1457-1466Crossref PubMed Google Scholar). In all fragments cysteine and methionine residues were replaced by amino acids with similar properties (Ser for Cys; Ile, Leu, and Val for Met residues) to retain feasibility of chemical cleavage and preserve functionality of the receptor as reported before (34.Cohen L.S. Becker J.M. Naider F. Biosynthesis of peptide fragments of eukaryotic GPCRs in Escherichia coli by directing expression into inclusion bodies.J. Pept. Sci. 2010; 16: 213-218PubMed Google Scholar, 35.Martin N.P. Celić A. Dumont M.E. Mutagenic mapping of helical structures in the transmembrane segments of the yeast α-factor receptor.J. Mol. Biol. 2002; 317: 765-788Crossref PubMed Scopus (33) Google Scholar). Several Escherichia coli expression strains (BL21(DE3), BL21-AI, BL21(DE3) pLysS, BL21(DE3) STAR pLysS, BL21 Rosetta pLysS, C41, and C43) were screened for expression (see “Experimental Procedures”). Inclusion bodies were solubilized in 70% TFA and, following chemical cleavage of the fusion proteins with CNBr, polypeptides of interest were purified using RP-HPLC. This resulted in expression yields of about 15 and 5 mg of purified protein per liter of 15N,13C-labeled and 15N,13C,2H-labeled M9 culture, respectively, for TM1 (using the strain BL21-AI), 11 and 5 mg/liters for TM123 (BL21-AI) (36.Caroccia K.E. Estephan R. Cohen L.S. Arshava B. Hauser M. Zerbe O. Becker J.M. Naider F. Expression and biophysical analysis of a triple-transmembrane domain-containing fragment from a yeast G protein-coupled receptor.Biopolymers. 2011; 96: 757-771Crossref PubMed Scopus (9) Google Scholar), and 15 and 5 mg/liters for TM127 (BL21(DE3)STARpLysS). Expression details of TM12 have been reported previously (37.Neumoin A. Cohen L.S. Arshava B. Tantry S. Becker J.M. Zerbe O. Naider F. Structure of a double transmembrane fragment of a G-protein-coupled receptor in micelles.Biophys. J. 2009; 96: 3187-3196Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). To obtain additional long-range distance restraints for TM127, the single cysteine mutants S47C, S75C, and S104C containing the full N terminus (Ser2-Thr114, Thr274-Leu340), and an N-terminal His10 tag separated by a cloning insertion coding for a C3 site (His10-Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Ser2 … ) were expressed. After RP-HPLC purification, 40 mg of the mutant proteins per liter of 15N-M9 medium culture were obtained. These sulfhydryl-containing mutants were successfully coupled to MTSL maleimide. For NMR measurements, samples were measured in 150 mm 1-palmitoyl-2-hydroxy-sn-glycero-3-(phospho-rac-(1-glycerol)) (LPPG)/dodecyl-phosphocholine (DPC) (4:1 mol/mol) micelles as the membrane mimetic, using 40 mm potassium phosphate buffer at pH 6.4. These conditions were similar to those previously used in our investigation of TM12 (37.Neumoin A. Cohen L.S. Arshava B. Tantry S. Becker J.M. Zerbe O. Naider F. Structure of a double transmembrane fragment of a G-protein-coupled receptor in micelles.Biophys. J. 2009; 96: 3187-3196Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The NMR samples of TM1, TM123, and TM127 all exhibit homogeneous line widths and good signal dispersion considering their highly α-helical nature (see Fig. 1 and supplemental Figs. S3–S6). Almost complete backbone assignment and partial side chain assignment could be achieved for every fragment (see below) using three-dimensional triple resonance as well as HCCH and 13C-resolved NOESY spectra. For TM1, 97% of the backbone and 66% of the side chain assignments were achieved. The assignments for TM12 as well as details of its structure calculation have been published previously (37.Neumoin A. Cohen L.S. Arshava B. Tantry S. Becker J.M. Zerbe O. Naider F. Structure of a double transmembrane fragment of a G-protein-coupled receptor in micelles.Biophys. J. 2009; 96: 3187-3196Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Samples containing TM123 exhibited a strong tendency to form soluble aggregates. The rate of aggregate formation depended on the detergent and the deuteration scheme; the TM123 sample with reverse ILV methyl labeling (completely perdeuterated protein with only the methyl groups of Ile, Leu, and Val residues protonated) aggregated completely within hours in deuterated detergents. Non-deuterated samples of the same polypeptide aggregated after several days. Aggregation was judged by the NMR line width and confirmed by size exclusion chromatography-multiangle light scattering (SEC-MALS) experiments (supplemental Fig. S2). Despite these challenges about 95% of the backbone resonances of TM123 could be assigned, the only missing residues being the two N-terminal amino acids, as well as two prolines (Pro79 and Pro117) and two residues at the beginning of TM2 (Asn84 and Gln85). Due to the high redundancy of certain amino acids and the resulting spectral overlap, as well as broad lines in the proton-carbon correlation spectra (HCCH-TOCSY and 13C-resolved NOESY) only 55% of side chains could be assigned. In contrast to TM123, TM127 did not aggregate in the micellar environment (supplemental Fig. S2) and it was possible to assign 93% of the backbone resonances and significantly more of the side chain resonances (63%) than for TM123. The two N-terminal amino acids and a region in the center of TM7 containing the Leu-Pro-Leu (residues 289–291) sequence were missing resulting in the slightly lower fraction of assigned backbone resonances despite the higher quality of the TM127 spectra compared with those of TM123. We suspect that conformational exchange in TM7 broadens these latter signals beyond detection. Selective reverse methyl labeling of Ile, Leu, and Val allowed us to assign most of the methyl groups but many of the remaining side chain atoms could not be uniquely assigned. As is often the case with membrane proteins, only a small number of these assignments yielded unambiguous NOE restraints. TM12 and TM127 or TM123 constitute two pairs of polypeptides in which nearly 80 residues are identical (those from TM12). We used these pairs to determine whether assignments on shorter fragments of GPCRs could be used to facilitate the assignments of the longer fragments. To transfer assignments, we compared corresponding strips in the three-dimensional 15N-resolved NOESY spectra of TM12 (37.Neumoin A. Cohen L.S. Arshava B. Tantry S. Becker J.M. Zerbe O. Naider F. Structure of a double transmembrane fragment of a G-protein-coupled receptor in micelles.Biophys. J. 2009; 96: 3187-3196Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) with those of TM123 and TM127 for residues from TM1 and TM2. The rationale behind this approach is that the amide protons of a given residue will be close to protons from its own side chain or from neighboring residues, but will likely not be close to side chain protons from other helices that form tertiary contacts. In fact, it is very rare to observe inter-chain NOEs for HN interactions with side chain protons of even closely packed helices because these are almost always more than 5 Å apart. Starting with cross-peaks in the TM123 or TM127, 15N,1H correlation spectra that were in the vicinity of an assigned peak in the TM12 correct assignment was derived from a peak pattern match of strips in the three-dimensional 15N-resolved NOESY spectrum. When using 15N,1H-HSQC and three-dimensional 15N-NOESY spectra were recorded at 900 MHz, 70 (93%) of the 75 strips of TM12 could be matched correctly for TM127. Similarly, 68 (90%) of the 75 strips of TM12 were successfully matched for the TM123 fragment. Details on this assignment procedure will be reported elsewhere. 4M. Poms, S. Jurt, P. Güntert, and O. Zerbe, unpublished data. Usually, structure calculations of helical membrane proteins suffer from an insufficient number of long-range restraints, partially due to the fact that complete side chain assignments are difficult to obtain. More importantly, suboptimal packing of helices in the not fully formed helix bundle, the fact that detergents are not a perfect mimic for biological membranes, and the inherent flexibility of membrane proteins result in exchange broadening that tends to damp out the weak but structurally important long-range NOEs. To compensate for the low number of NOE restraints, residual dipolar couplings (RDCs), paramagnetic relaxation enhancements (PREs), and chemical shift-derived restraints were used. We have also probed access to a water-soluble spin label to reveal which residues are solvent exposed. All these data were used to obtain restraints for the final structure calculation and to orient the fragments in the micelles. To judge how well primary NMR data were represented by the ensemble, back-prediction of the raw data from conformers of the NMR ensemble was carried out. Based on TALOS-N (38.Shen Y. Bax A. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks.J. Biomol. NMR. 2013; 56: 227-241Crossref PubMed Scopus (717) Google Scholar), backbone chemical shift data were used to predict the propensity of regions of Ste2p to form helices (Fig. 2). In general, all putative helices seem to be in the regions that are predicted by hydropathy algorithms. However, in the single TM fragment TM1 is clearly destabilized in the center of the helix around a GXXXG (GVRSG, residues 56–60) motif. In contrast this same region is significantly rigidified upon packing against TM2 in TM12. Accordingly the largest chemical shift differences between TM1 and TM2 for the overlapping segment are observed for the GVRSG residues (supplemental Fig. S8). The rigidification of the GVRSG region in TM1 is also observed in TM123 and TM127. Despite this rigidification there is a small but reproducible destabilization of the TM1 helices in the GVRSG domain also in the longer constructs. Based on the TALOS predictions the N-terminal end of the TM2 helix is destabilized in TM12, TM123, and TM127. In the latter polypeptide some assignments are missing, possibly indicating the presence of conformational exchange. 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• W2554301698 modified "2023-09-26" @default.
• W2554301698 title "NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates" @default.
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