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• W2040476959 abstract "The visual photoreceptor rhodopsin is a prototypical class I (rhodopsin-like) G protein-coupled receptor. Photoisomerization of the covalently bound ligand 11-cis-retinal leads to restructuring of the cytosolic face of rhodopsin. The ensuing protonation of Glu-134 in the class-conserved D(E)RY motif at the C-terminal end of transmembrane helix-3 promotes the formation of the G protein-activating state. Using transmembrane segments derived from helix-3 of bovine rhodopsin, we show that lipid protein interactions play a key role in this cytosolic “proton switch.” Infrared and fluorescence spectroscopic pKa determinations reveal that the D(E)RY motif is an autonomous functional module coupling side chain neutralization to conformation and helix positioning as evidenced by side chain to lipid headgroup Foerster resonance energy transfer. The free enthalpies of helix stabilization and hydrophobic burial of the neutral carboxyl shift the side chain pKa into the range typical of Glu-134 in photoactivated rhodopsin. The lipid-mediated coupling mechanism is independent of interhelical contacts allowing its conservation without interference with the diversity of ligand-specific interactions in class I G protein-coupled receptors. The visual photoreceptor rhodopsin is a prototypical class I (rhodopsin-like) G protein-coupled receptor. Photoisomerization of the covalently bound ligand 11-cis-retinal leads to restructuring of the cytosolic face of rhodopsin. The ensuing protonation of Glu-134 in the class-conserved D(E)RY motif at the C-terminal end of transmembrane helix-3 promotes the formation of the G protein-activating state. Using transmembrane segments derived from helix-3 of bovine rhodopsin, we show that lipid protein interactions play a key role in this cytosolic “proton switch.” Infrared and fluorescence spectroscopic pKa determinations reveal that the D(E)RY motif is an autonomous functional module coupling side chain neutralization to conformation and helix positioning as evidenced by side chain to lipid headgroup Foerster resonance energy transfer. The free enthalpies of helix stabilization and hydrophobic burial of the neutral carboxyl shift the side chain pKa into the range typical of Glu-134 in photoactivated rhodopsin. The lipid-mediated coupling mechanism is independent of interhelical contacts allowing its conservation without interference with the diversity of ligand-specific interactions in class I G protein-coupled receptors. G protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCRG protein-coupled receptorCHcholesterolDMn-dodecyl β-d-maltosidedansyl-PE1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl)FRETFörster resonance energy transferFTIRFourier transform infraredPCl-α-phosphatidylcholinePS1,2-dipalmitoyl-sn-glycero-3-phospho-l-serineOGn-octyl β-d-glucopyranoside. are hepta-helical membrane proteins that couple a large variety of extracellular signals to cell-specific responses via activation of G proteins. In the visual photoreceptor rhodopsin, a prototypical class I GPCR (1Mustafi D. Palczewski K. Mol. Pharmacol. 2009; 75: 1-12Crossref PubMed Scopus (89) Google Scholar, 2Weis W.I. Kobilka B.K. Curr. Opin. Struct. Biol. 2008; 18: 734-740Crossref PubMed Scopus (77) Google Scholar), molecular activation processes can be monitored in real time by spectroscopic assays and analyzed in the context of several crystal structures (3Salom D. Lodowski D.T. Stenkamp R.E. Le Trong I. Golczak M. Jastrzebska B. Harris T. Ballesteros J.A. Palczewski K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16123-16128Crossref PubMed Scopus (394) Google Scholar, 4Okada T. Fujiyoshi Y. Silow M. Navarro J. Landau E.M. Shichida Y. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 5982-5987Crossref PubMed Scopus (657) Google Scholar, 5Palczewski 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. Science. 2000; 289: 739-745Crossref PubMed Scopus (5035) Google Scholar, 6Li J. Edwards P.C. Burghammer M. Villa C. Schertler G.F. J. Mol. Biol. 2004; 343: 1409-1438Crossref PubMed Scopus (674) Google Scholar, 7Park J.H. Scheerer P. Hofmann K.P. Choe H.W. Ernst O.P. Nature. 2008; 454: 183-187Crossref PubMed Scopus (808) Google Scholar, 8Scheerer P. Park J.H. Hildebrand P.W. Kim Y.J. Krauss N. Choe H.W. Hofmann K.P. Ernst O.P. Nature. 2008; 455: 497-502Crossref PubMed Scopus (929) Google Scholar). The primary signal for rhodopsin is the 11-cis to all-trans photoisomerization of retinal covalently bound to the apoprotein opsin through a protonated Schiff base to Lys296. Current models converge toward a picture in which “microdomains” act as conformational switches that are coupled to different degrees to the primary activation process. Two activating “proton switches” have been identified (9Vogel R. Sakmar T.P. Sheves M. Siebert F. Photochem. Photobiol. 2007; 83: 286-292Crossref PubMed Scopus (29) Google Scholar) as follows: breakage of an intramolecular salt bridge (10Rao V.R. Oprian D.D. Annu. Rev. Biophys. Biomol. Struct. 1996; 25: 287-314Crossref PubMed Scopus (135) Google Scholar) by transfer of the Schiff base proton to its counter ion Glu-113 (11Jäger F. Fahmy K. Sakmar T.P. Siebert F. Biochemistry. 1994; 33: 10878-10882Crossref PubMed Scopus (146) Google Scholar) is followed by movement of helix-6 (H6) (12Altenbach C. Kusnetzow A.K. Ernst O.P. Hofmann K.P. Hubbell W.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 7439-7444Crossref PubMed Scopus (379) Google Scholar, 13Kusnetzow A.K. Altenbach C. Hubbell W.L. Biochemistry. 2006; 45: 5538-5550Crossref PubMed Scopus (76) Google Scholar) in the metarhodopsin IIa (MIIa) to MIIb transition. The MIIb state takes up a proton at Glu-134 (14Arnis S. Fahmy K. Hofmann K.P. Sakmar T.P. J. Biol. Chem. 1994; 269: 23879-23881Abstract Full Text PDF PubMed Google Scholar) in the class-conserved D(E)RY motif at the C-terminal end of helix-3 (H3) leading to the MIIbH+ intermediate (15Knierim B. Hofmann K.P. Ernst O.P. Hubbell W.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 20290-20295Crossref PubMed Scopus (113) Google Scholar, 16Vogel R. Mahalingam M. Lüdeke S. Huber T. Siebert F. Sakmar T.P. J. Mol. Biol. 2008; 380: 648-655Crossref PubMed Scopus (136) Google Scholar), which activates transducin (Gt), the G protein of the photoreceptor cell. Glu-134 regulates the pH sensitivity of receptor signaling (17Fahmy K. Sakmar T.P. Biochemistry. 1993; 32: 7229-7236Crossref PubMed Scopus (135) Google Scholar) in membranes as reviewed previously (18Lüdeke S. Mahalingam M. Vogel R. Photochem. Photobiol. 2009; 85: 437-441Crossref PubMed Scopus (19) Google Scholar), and in complex with Gt the protonated state of the carboxyl group becomes stabilized (19Fahmy K. Sakmar T.P. Siebert F. Biochemistry. 2000; 39: 10607-10612Crossref PubMed Scopus (74) Google Scholar). This charge alteration is linked to the release of an “ionic lock,” originally described for the β2-adrenergic receptor (20Ballesteros J.A. Jensen A.D. Liapakis G. Rasmussen S.G. Shi L. Gether U. Javitch J.A. J. Biol. Chem. 2001; 276: 29171-29177Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar), which also in rhodopsin stabilizes the inactive state (16Vogel R. Mahalingam M. Lüdeke S. Huber T. Siebert F. Sakmar T.P. J. Mol. Biol. 2008; 380: 648-655Crossref PubMed Scopus (136) Google Scholar) through interactions between the cytosolic ends of H3 and H6 (21Sheikh S.P. Zvyaga T.A. Lichtarge O. Sakmar T.P. Bourne H.R. Nature. 1996; 383: 347-350Crossref PubMed Scopus (398) Google Scholar). G protein-coupled receptor cholesterol n-dodecyl β-d-maltoside 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl) Förster resonance energy transfer Fourier transform infrared l-α-phosphatidylcholine 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine n-octyl β-d-glucopyranoside. In the absence of a lipidic bilayer, proton uptake and H6 movement become uncoupled (15Knierim B. Hofmann K.P. Ernst O.P. Hubbell W.L. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 20290-20295Crossref PubMed Scopus (113) Google Scholar). Lipidic composition affects MII formation, rhodopsin structure, and oligomerization (22Gibson N.J. Brown M.F. Biochemistry. 1993; 32: 2438-2454Crossref PubMed Scopus (163) Google Scholar, 23Botelho A.V. Huber T. Sakmar T.P. Brown M.F. Biophys. J. 2006; 91: 4464-4477Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 24Soubias O. Niu S.L. Mitchell D.C. Gawrisch K. J. Am. Chem. Soc. 2008; 130: 12465-12471Crossref PubMed Scopus (63) Google Scholar) and differs at the rhodopsin membrane interface from the bulk lipidic phase (25Soubias O. Teague W.E. Gawrisch K. J. Biol. Chem. 2006; 281: 33233-33241Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Likewise, MII formation specifically affects lipid structure (26Isele J. Sakmar T.P. Siebert F. Biophys. J. 2000; 79: 3063-3071Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Although of fundamental importance for GPCR activation, the potential implication of lipid protein interactions in “proton switching” is not clear. A functional role of Glu-134 in lipid interactions has been originally derived from IR spectra where E134Q replacement abolished changes of lipid headgroup vibrations in the MIIGt complex (19Fahmy K. Sakmar T.P. Siebert F. Biochemistry. 2000; 39: 10607-10612Crossref PubMed Scopus (74) Google Scholar). Computational approaches emphasized the “strategic” location of the D(E)RY motif (27Huber T. Botelho A.V. Beyer K. Brown M.F. Biophys. J. 2004; 86: 2078-2100Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), and the Glu-134 carboxyl pKa may critically depend on the lipid protein interface (28Periole X. Ceruso M.A. Mehler E.L. Biochemistry. 2004; 43: 6858-6864Crossref PubMed Scopus (21) Google Scholar). However, the implications for proton switching are not evident, and the theoretical interest is contrasted by the lack of experimental data addressing the effect of the lipidic phase on side chain protonation, secondary structure, and membrane topology of the D(E)RY motif. We have studied the coupling between conformation and protonation in single transmembrane segments derived from H3 of bovine rhodopsin. We have assessed the “modular” function of the D(E)RY motif by determining parameters not evident from the crystal structures, i.e. the pKa of the conserved carboxyl, its linkage to helical structure, and the effect of protonation on side chain to lipid headgroup distance. We show that the D(E)RY motif encodes an autonomous “proton switch” controlling side chain exposure and helix formation in the low dielectric of a lipidic phase. The data ascribe a functional role to lipid protein interactions that couple the chemical potential of protons to an activity-promoting GPCR conformation in a ligand-independent manner. 1,2-Dipalmitoyl-sn-glycero-3-phospho-l-serine (PS), l-α-phosphatidylcholine (PC), cholesterol (CH), n-octyl β-d-glucopyranoside (OG), and n-dodecyl β-d-maltoside (DM) were from Sigma. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl) (dansyl-PE) was from Avanti Polar Lipids (Alabaster, AL). Fourier transform IR (FTIR) spectra were obtained with peptides (∼10 mg/ml) in 5% DM, 100 mm sodium phosphate buffer, using a vector 22 spectrometer (Bruker, Ettlingen, Germany) at 2 cm−1 resolution. 30 μl of the sample were transferred into a Bio-ATR-II cell (Bruker, Ettlingen, Germany) and the pH changed from 8.8 to 3 by dialysis (100 mm sodium phosphate). Pure buffer spectra were used as spectral references to calibrate the pH-dependent absorption strength and frequency of the phosphate stretching modes. Therefore, a very sensitive real time monitor of pH changes could be implemented. Circular dichroism spectra were recorded at room temperature with a J-815 instrument (Jasco, Gross-Umstadt, Germany) at ∼4 liters/min N2 flow rate from 200 to 260 nm in 0.1-cm cuvettes on DM-solubilized peptides (0.2–0.3 mg/ml). Ellipticity (θ) was recorded in millidegrees. Fluorescence measurements were performed with an LS55 spectrometer (PerkinElmer Life Sciences) in 200-μl cuvettes at 285 nm for Trp excitation and emission recorded from 290 to 450 nm (slit width of 5 nm). Peptide-containing micelles were prepared by solubilization of 1–2 mg of peptide in ∼200 μl of 5% DM followed by 30-fold dilution in 100 mm phosphate buffer of different pH. “Lipid-doped” peptide-containing micelles were prepared by dissolving ∼2 mg of peptide in 5% DM (∼600 μl) and mixing with the appropriate vacuum-dried amounts of dansyl-PE and PS in a molar ratio of 1:2.5:1, respectively. 20 μl aliquots were diluted 30-fold in a series of phosphate buffers (100 mm) allowing duplicate recording of emission spectra at each pH. Peptide-containing vesicles were prepared by mixing 1–2 mg of peptide solubilized in ∼ 800 μl of 5% OG with PS, dansyl-PE, and PC in the ratio 1:2.5:2.5, respectively, with a final total lipid:peptide ratio of 50–80. The detergent was removed by 15 h of flow cell dialysis, and the suspension was sonicated and freeze-thawed 10 times and diluted in 100 mm phosphate buffer. Cholesterol-containing vesicles were prepared in parallel from the same stock by supplementing an aliquot of the OG-solubilized mixture with cholesterol to a final total lipid:cholesterol ratio of 1:0.2. Peptides were synthesized and high pressure liquid chromatography-purified (free of trifluoroacetate) by ThermoFisher (Ulm, Germany) with C and N termini amidated and acetylated, respectively. Peptides of the following sequences were derived from amino acids 108–138 in H3 of rhodopsin: H3E, TGC NLA(E) GFF ATL GGA(E) IAL WSL VVL AIE RYV V; H3E27Q, TGC NLA(E) GFF ATL GGA(E) IAL WSL VVL AIQ(E) RYV V; H3E27D, TGC NLA(E) GFF ATL GGA(E) IAL FSL VVL AID RYV W; H3W19F/V31W, TGC NLA(E) GFF ATL GGA(E) IAL F(W)SL VVL AIE RYV W(V); H3W19F/V31W/E27Q, TGC NLA(E) GFF ATL GGA(E) IAL F(W)SL VVL AIQ(E) RYV W(V); native amino acids in parentheses were replaced by the preceding ones. The helicity of the peptides was assessed by infrared spectroscopy and circular dichroism measurements. As shown in Fig. 1, the degree of helicity depends strongly on even very subtle changes in the sequence. In the context of the most native-like sequence in peptide H3E, the additional shift of the tryptophan from position 19 to 31 as well as the further replacement of the Glu-134 homologue by glutamine does not interfere with a predominantly helical secondary structure (inset). Spectral decomposition of the integral IR absorption in the amide I range into gaussian/lorentzian curves shows 70–80% α-helical structure for H3E and H3W19F/V31W/E27Q with peak frequencies at 1654–1656 cm−1. The CD spectra of the latter peptides (Fig. 1, inset) were converted to mean residue ellipticity in degrees·dmol−1·cm−2 as θres = 100·θ/(c·l·n), where c is the molar concentration of the peptide; l is optical path length (0.1 cm), and n is the number of amino acids in the peptide (here 31). The fraction p of helical structure was calculated as p = −θres222/(39,500·(1–2.57/ν)) as described (29Chen Y.H. Yang J.T. Chau K.H. Biochemistry. 1974; 13: 3350-3359Crossref PubMed Scopus (1964) Google Scholar). Consistency requires that the average number ν of peptide bonds in helical conformation complies with ν/31 = p, which was obtained for ν = 22 and p = 0.71. Thus, ∼22 out of the total of 31 amino acids in each of the two peptides are on average in a helical conformation that is on the lower side of the range estimated by FTIR spectroscopy and suggests that 4–5 disordered amino acids form the C- and N-terminal helical ends. In contrast to the largely helical peptides H3W19F/V31W and H3W19F/V31W/E27Q, the related pair of sequences H3E and H3E27Q shows helicity only for H3E. Three independent synthesis rounds using different technologies failed to produce peptides H3E27Q and H3E27D with helical structures upon detergent solubilization. The CD spectra of these peptides are rather featureless (Fig. 1, inset), and their IR absorption shows only a small fraction of helical structure (1656 cm−1) relative to a predominating amide I mode at low frequency indicative of aggregation. Thus, under the conditions of the FTIR and CD experiments, the replacement of Glu-27 by Gln allows helix formation only in the presence of the additional W19F replacement. Therefore, the proton-induced conformational switch was studied by FTIR in the least modified sequence, i.e. H3E, whereas the negative control was provided by H3W19F/V31W/E27Q. For ICL2, AIE RYV W(V)VC KPM SNF RFG, this peptide was derived from amino acids 132–149, containing the C-terminal end of H3 and its cytosolic extension. The linkage between conformation and carboxyl protonation in the D(E)RY motif was studied in the peptide H3E corresponding to amino acids 108 to 138 of rhodopsin. The native residues Glu-113 and Glu-122 were replaced by alanines, rendering the carboxylate in the Glu-134 homologue (i.e. Glu-27 in the peptide) the only carboxyl in H3E. The symmetric carboxylate stretching mode of Glu-27 (1401 cm−1) and the narrow amide I absorption at 1656 cm−1 (typical of the helical secondary structure of the peptide backbone) were used to monitor by FTIR spectroscopy the side chain protonation and secondary structure, respectively. Fig. 2A shows the absolute IR absorption and the pH-induced difference spectra, obtained by dialysis-coupled attenuated total reflectance-FTIR difference spectroscopy (30Fahmy K. Recent Research Developments in Biophysical Chemistry.in: Pandalai S.G. Vol. 2. Research Sign post, Trivandrum, Kerala, India2001: 1-17Google Scholar). Spectra recorded at different pH values were subtracted from a reference spectrum acquired at pH 8.8, where the Glu-134 homologue is fully ionized (maximal absorption at 1401 cm−1). The stretching vibrations of the buffer phosphates between 1200 and 800 cm−1 served as a pH monitor. The macroscopic pKa of Glu-27 was identified by the spectrum with half-maximal intensity of the 1401 cm−1 band. The latter was obtained at pH 5.9, i.e. 1–2 pH units above the pK of a glutamate side chain in aqueous solution. Also the peptide backbone exhibited pH-dependent transitions, giving rise to changes of the amide II band around 1550 cm−1 (overlapping with the antisymmetric carboxylate stretching mode at ∼1560 cm−1) and the amide I absorption at 1656 cm−1. The structural change was evaluated using the titration spectra in Fig. 2B. The inset in Fig. 2B shows that the 1656/1638 cm−1 absorption change correlates linearly with the carboxylate absorption at 1401 cm−1. The secondary structural change is coupled to side chain protonation with a common pKa of 5.9, with a more helical structure favored at acidic pH. The integral intensity change at 1656 cm−1 corresponds to 3–5% of the total amide I absorption of H3E (Fig. 2A), indicating that side chain protonation extends the helical structure by an average of 1–2 peptide bonds. This process was abolished upon E27Q replacement in the peptide H3W19F/V31W/E27Q (readily adopting a helical structure in contrast to the alternative negative control sequence H3E27Q, see “Experimental Procedures”), which did not exhibit the pH-sensitive 1656/1638 cm−1 difference band (Fig. 2B). Thus, the H3 secondary structure is controlled by the protonation state of the carboxyl in the D(E)RY motif. The functionality of the proton-dependent rearrangement of the C-terminal structure in the H3W19F/V31W background containing the Glu-134 homologue is shown below and proves its independence from the altered location of the Trp. The upshift of the pKa relative to that of a glutamate in water indicates that side chain protonation at the H3 C terminus is stabilized by the concomitant helix extension. To prove the localization of the structural transition, a Trp was C-terminally attached, replacing the Val-138 homologue of rhodopsin, and for the purpose of specificity, Trp-19 (corresponding to the native Trp-126) was replaced by Phe, resulting in the construct H3W19F/V31W, which shows a pH-sensitive emission in the 300–400 nm range (Fig. 3). Trp-31 is two residues away from the tyrosine of the D(E)RY motif. It acts as an energy acceptor for the excited state of tyrosine, which shows appreciable emission only in the absence of Trp-31 or in the presence of the more distant Trp-19 (Fig. 3, inset A). The Trp-dominated emission in H3W19F/V31W provides a sensitive fluorescence monitor of structural transitions in the D(E)RY motif due to both the geometrical constraints for FRET and the hydrophobicity dependence of Trp emission itself. The pH-induced emission difference spectra show that in the ionized state of Glu-27 (positive lobe at 365 nm) the emission of Trp-31 is shifted to longer wavelengths versus the protonated state (negative lobe at 320 nm). This could reflect reduced quenching of tyrosine as well as exposure of Trp to a more hydrophobic environment at pH <6. The half-maximal emission change from the D(E)RYVW sequence is interpolated to pH 5.6 and is lost upon E27Q replacement. This parallels the infrared results and supports the localization of the detected secondary structure formation within the direct vicinity of the D(E)RY motif. The protonation-induced blue shift of the Trp emission maximum is also observed with H3W19F/V31W in DOPC vesicles (Fig. 3), although with an ∼50% reduced amplitude that is probably caused by the presence of zwitterionic (i.e. more hydrophilic) headgroups as compared with the neutral phase boundary of the DM micelle. Due to the strong light scatter in the near-UV light caused by the vesicles, the signal to noise ratio is also ∼5-fold reduced. In contrast to the systematic pH dependence of Trp emission shown for the TM3-derived peptides, an unsystematic pH dependence was obtained from the peptide ICL2 (Fig. 3, inset B) containing the D(E)RY motif and the ensuing second cytosolic loop of rhodopsin (Ala-132 to Gly-149 with V138W replacement). Side chain neutralization and helix extension in the vicinity of the D(E)RY motif is expected to alter the hydrophobic length of the H3 segment. To test whether these coupled processes affect the distance between the H3 C terminus and the phase boundary in a micellar or lipidic environment, the C-terminal Trp of the peptide H3W19F/V31W was employed as a donor fluorphore whose emission is quenched by a dansyl group (via FRET) in measurements, including dansyl-labeled phosphatidylethanolamine (dansyl-PE). Fig. 4 (inset) shows that excitation at 285 nm of “dansyl-PE-doped” peptide-containing micelles evokes emission from Trp and dansyl at 345 and 524 nm, respectively. Trp emission was typically 10–30% of that in the absence of dansyl-PE. The 524 nm emission increased upon acidification, whereas Trp emission decreased, indicating a more efficient energy transfer when Glu-27 is protonated. dansyl absorption and emission are intrinsically pH-independent in the pH 4–10 range (31Lowe M.P. Parker D. Inorg. Chim. Acta. 2001; 317: 163-173Crossref Scopus (53) Google Scholar, 32Koike T. Watanabe T. Aoki S. Kimura E. Shiro M. J. Am. Chem. Soc. 1996; 118: 12696-12703Crossref Scopus (263) Google Scholar). To correct for the different amounts of dansyl-PE-labeled micelles or vesicles in the different samples, however, the residual Trp emission was normalized with respect to the fluorescence efficiency upon direct excitation of dansyl-PE at 336 nm in these systems. Therefore, the residual Trp emission from samples derived from the same stock of a given lipidic composition but transferred to different buffers is scaled to the same total concentration of vesicles in each buffer. The evaluation is restricted to the quenching of the donor fluorescence, i.e. to the residual Trp emission at 345 nm. This allows comparing the pH sensitivity of dansyl-mediated quenching of Trp-31 emission in micelles and vesicles consisting of PC, PS, and cholesterol at different pH values (Fig. 4) and independently of the determination of absolute FRET efficiency. For better comparison of the apparent pKa values obtained from fits to Henderson-Hasselbach curves, the traces (and original data) were scaled to the fitted signal level at pH 9 for each sample. The quenching of Trp-31 exhibited an apparent pKa of 6 ± 0.3, which was independent of the assessed lipid composition and of the presence of cholesterol within the accuracy of the experiment. Trp fluorescence was more efficiently quenched at acidic pH indicating that upon neutralization of Glu-27 the C terminus moves from an aqueous to a more hydrophobic environment near the lipid headgroups. This agrees with the slight blue shift of the Trp emission (causing a negligible change of the overlap integral between dansyl absorption and the Trp emission of <0.5%) and would favor the deactivation of the excited state of Trp-31 upon shortening of the averaged donor to acceptor distance. Alternatively, the C-terminal Trp may become rotationally constrained if it gets partially immersed in the lipidic phase. This could also reduce Trp emission if a more favorable alignment with the dansyl electronic transition moment is achieved. The same mechanistic conclusion would apply; the H3W19F/V31W C terminus moves from an exposed to a more lipid-immersed state with Glu-27 protonated, whereas the opposite transition contradicts the data. No attempt was made to determine and compare absolute FRET efficiencies in the different lipidic compositions by further evaluation of the dansyl emission at 524 nm in relation to Trp quenching. The unknown effect on the rotational freedom of both the C-terminal Trp and the dansyl group in the different lipidic compositions would render a more detailed analysis ambiguous. The high pKa of carboxyl protonation in micelles and vesicles implies that the protonated state of the Glu-134 couples to a free enthalpy-delivering reaction. The coupling depends on the α-helix preceding the D(E)RY motif in a hydrophobic phase as it is not seen in ICL2 (Fig. 3). The low dielectric constant in vesicle membranes and in the interior of a micelle could promote both stabilization of the protonated side chain and of helical secondary structure. Therefore, we have asked whether a hydrophobicity-mediated proton switch is consistent with thermodynamic estimates of helix stability and charge-dependent partitioning of a carboxyl side chain at a phase boundary. The coupling of conformation to protonation is described by the microscopic equilibria defined in Fig. 5A. Peptide conformations with an exposed (E) or buried (B) state of the ionized carboxylate of Glu-27 interconvert with the equilibrium constant KC and their protonated counterparts (EH and BH) with the equilibrium constant KCH. State function properties require that the free enthalpy difference for the conformational transition in the protonated versus the ionized state (i.e. ΔΔG = −RT ln KCH/KC) equals that of transferring a neutral versus an ionized glutamic acid side chain from an aqueous to a hydrophobic medium (i.e. ΔΔG = −RT ln KB/KE). Free enthalpies ΔGtr have been determined for water to octanol transfers (33Wimley W.C. Creamer T.P. White S.H. Biochemistry. 1996; 35: 5109-5124Crossref PubMed Scopus (475) Google Scholar). The reported ΔΔG of 14.7 kJ (3.52 kcal) for glutamic acid provides an estimate of the enthalpy available to link protonation to conformation by a purely hydrophobicity-driven mechanism. These transfer enthalpies do indeed reproduce the measured pKa of 5.9 for protonation/helix extension with a ΔGc of 6 kJ for the transition of the ionized state from the exposed to the buried conformation. The resulting free energy surface of the system is shown in Fig. 5B. Upon acidification, the system moves along the “valley” from less than 10% protonation with >80% of the carboxylates exposed to the aqueous phase at pH 7 (microscopic pKB of the buried state) to more than 90% protonation with >80% of the neutral side chains buried at pH 4.4 (microscopic pKE of the exposed state). The equilibrium states trace out a trajectory that runs roughly diagonally through the conformation/protonation plane reproducing the linearity between the measured amide I and carboxylate absorption changes. Breakage of the ionic lock at the cytosolic H3/H6 interface in the transition to the Gt-activating state of rhodopsin is linked to protonation of Glu-134 in the class-conserved D(E)RY motif in H3. This constitutes one of the two proton switches that control rhodopsin activation (34Mahalingam M. Martínez-Mayorga K. Brown M.F. Vogel R. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 17795-17800Crossref PubMed Scopus (131) Google Scholar) and is likely to operate in other class I GPCRs (20Ballesteros J.A. Jensen A.D. Liapakis G. Rasmussen S.G. Shi L. Gether U. Javitch J.A. J. Biol. Chem. 2001; 276: 29171-29177Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, 35Shukla A.K. Sun J.P. Lefkowitz R.J. Mol. Pharmacol. 2008; 73: 1333-1338Crossref PubMed Scopus (30) Google Scholar, 36Rosenbaum D.M. Cherezov V. Hanson M.A. Rasmussen S.G. Thian F.S. Kobilka T.S. Choi H.J. Yao X.J. Weis W.I. Stevens R.C. Kobilka B.K. Science. 2007; 318: 1266-1273Crossref PubMed Scopus (1189) Google Scholar, 37Scheer A. Fanelli F. Costa T. De Benedetti P.G. Cotecchia S. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 808-813Crossref PubMed Scopus (200) Google Scholar, 38Greasley P.J. Fanelli F. Rossier O. Abuin L. Cotecchia S. Mol. Pharmacol. 2002; 61: 1025-1032Crossref PubMed Scopus (107) Google Scholar). Here we have shown that the D(E)RY motif represents an autonomous proton switch that is not per se dependent on interhelical contacts. Instead, local lipid protein interactions alter the pKa of the side chain carboxylate by providing a medium of low dielectricity that stabilizes the" @default.
• W2040476959 created "2016-06-24" @default.
• W2040476959 creator A5051546849 @default.
• W2040476959 creator A5065204390 @default.
• W2040476959 date "2009-10-01" @default.
• W2040476959 modified "2023-10-16" @default.
• W2040476959 title "Lipid Protein Interactions Couple Protonation to Conformation in a Conserved Cytosolic Domain of G Protein-coupled Receptors" @default.
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