Báo cáo khoa học: Conformational heterogeneity of transmembrane residues after the Schiff base reprotonation of bacteriorhodopsin 15 docx

Conformational heterogeneity of transmembrane residues after the Schiff base reprotonation of bacteriorhodopsin 15 N CPMAS NMR of D85N/ T170C membranes A James Mason1, George J Turner2 and Clemens Glaubitz1 Centre for Biomolecular Magnetic Resonance and Institut fur Biophysikalische Chemie, J.W Goethe Universitat, Frankfurt, Germany ă ă Department of Chemistry and Biochemistry, Seton Hall University, South Orange, NJ, USA Keywords bacteriorhodopsin; solid-state NMR; N-state; O-state Correspondence C Glaubitz, Institut fur Biophysikalische ă Chemie, Centre for Biomolecular Magnetic Resonance, J.W Goethe Universitat, Marieă Curie Str 9, D-60439 Frankfurt, Germany Fax: +49 69798 29929 Tel: +49 69798 29927 E-mail: glaubitz@chemie.uni-frankfurt.de (Received 19 October 2004, revised 10 February 2005, accepted 28 February 2005) bR, N-like and O-like intermediate states of [15N]methionine-labelled wild type and D85N ⁄ T170C bacteriorhodopsin were accumulated in native membranes by controlling the pH of the preparations 15N cross polarization and magic angle sample spinning (CPMAS) NMR spectroscopy allowed resolution of seven out of nine resonances in the bR-state It was possible to assign some of the observed resonances by using 13C ⁄ 15N rotational echo double resonance (REDOR) NMR and Mn2+ quenching as well as D2O exchange, which helps to identify conformational changes after the bacteriorhodopsin Schiff base reprotonation The significant differences in chemical shifts and linewidths detected for some of the resonances in N- and O-like samples indicate changes in conformation, structural heterogeneity or altered molecular dynamics in parts of the protein doi:10.1111/j.1742-4658.2005.04633.x Bacteriorhodopsin [1] is a 26 kDa seven transmembrane helix protein (7TM) found in the extremely halophilic archeaon Halobacterium salinarium [2] The proton pumping ability of this protein is conferred by the prosthetic retinal attached via a Schiff base to Lys216 The light-induced isomerization from all-trans to 13-cis causes the release of a proton from the Schiff base, which in turn causes a proton to be released at the extracellular surface The reaction is cyclic and the photocycle has been characterized spectroscopically where a series of photointermediates have been determined: bR570 ! K590 ! L550 ! M412 ! N560 ! O640 ! bR570 The photocycle can be divided into two phases The first phase is the K-L-M1-M2-M2¢ sequence, where a proton is donated from the Schiff base to Asp85 and another proton is released to the extracellular surface, and the second is the N-N¢-O-bR sequence, where the Schiff base is reprotonated from Asp96 Asp96 is itself reprotonated from the cytoplasmic surface and a proton is transferred from Asp85 to the proton release site Analysis of the photomechanism has been revolutionized by the production of a family of high resolution X-ray diffraction structures [3,4] The structures and structural changes assigned to the intermediates of reprotonation reactions remain an area of debate, as described below N-state In the early intermediates of the reprotonation phase, when the protein is in the late M- and N-state, contrasting measurements of the movements in the Abbreviations 7TM, seven transmembrane helix protein; CPMAS, cross polarization and magic angle sample spinning; DA, dark adapted; LA, light adapted; REDOR, rotational echo double resonance 2152 FEBS Journal 272 (2005) 2152–2164 ª 2005 FEBS A J Mason et al cytoplasmic half of the protein have been obtained Large scale motions have been observed, particularly in helices E, F and G, and the EF loop, by a variety of techniques, including electron diffraction [5–7], X-ray diffraction in projection of purple membranes [8–10] and Electron Spin Resonance (ESR) spin labelling [11,12] An early X-ray diffraction study of F171C ˚ membranes [13], at A resolution, observed fairly small structural changes with the largest change involving a movement of helix F and some small movements of helices B and G, whilst two electron diffraction studies of 2D crystals observed rather large structural changes in the cytoplasmic region The structure of a ‘cytoplasmically open’ conformation found in the D96G ⁄ F171C ⁄ F219L triple mutant [6] revealed displacements of the ends of helices F and G ˚ of 3.5 and A, respectively, while the structure of the N intermediate found in F219L membranes [7] showed ˚ that both helices E and F are displaced by some A, with helix G again moving slightly These results were in contrast, however, with high resolution structures of the MN- and N¢-states [14,15], produced from 3D crystals, which not show the expected tilts or rotations [16] It has been suggested that, within 3D crystals, the crystal lattice resists any increase in the unit cell dimensions preventing such conformational changes O-state The bacteriorhodopsin O-state is the least well resolved conformer of the reprotonation mechanism The most recent analysis relies on the mutants D85S and D85S ⁄ F219L as O-state models [17] The structures of ˚ D85S and D85S ⁄ F219L, at 2.25 and 2.0 A resolution respectively, reveal important differences between the bR- and O-like states [17] The most notable differences are in the extracellular half of the protein and in the loop regions, particularly the BC, DE and EF loops A slight repackaging of the transmembrane helices in the extracellular side results in tilting of the helices A, B, C and D by approximately 3° and, more noticeably, helix E by 6.9° relative to the bR-state The protonation state of Asp85 plays a central role in the conformational changes and linked proton movements during the transitions between the M-, N-, O- and bR-states As illustrated in the discussion of the O-state models, mutants of Asp85 have been useful in the study of the reprotonation mechanism [17] Replacement of Asp85 with asparagine (D85N) allows study of the intermediate state conformations in which Asp85 is normally protonated The bR mutant D85N exists as three spectrally distinct species in a pHdependent equilibrium [18] Transitions between these FEBS Journal 272 (2005) 2152–2164 ª 2005 FEBS N- and O-states of BR seen by solid-state NMR species regulate the pKa values of Asp96 and the Schiff base in a manner consistent with that observed in the reprotonation phase of the wild-type protein At lowto-neutral pH an O-like species predominates (kmax ¼ 615 nm), whereas at higher pH values increasing levels of an N- (kmax ¼ 570 nm) and an M-like species (kmax ¼ 410 nm) appear [18] D85N, and second site mutants thereof, can be used to isolate the conformational transitions of the reprotonation phase of proton pumping In this study we exploited the pH-dependent transitions of the D85N ⁄ T170C double mutant to probe the structures of N- and O-like states Our mutant D85N ⁄ T170C behaves similarly to D85N [18] However, the pKa of M accumulation is raised by the additional cysteine mutation and hence, although some M-state remains, it is less populated making this system more suitable to access N-and O-like states [19] We applied residue-specific 15N labelling to all methionines in the wild type purple membrane (Fig 1) and D85N ⁄ T170C membrane to evaluate the conformational flexibility of transmembrane helices in the bR, 163 32 20 145 117 56 208 54 118 209 60 68 Fig The three-dimensional structure of bacteriorhodopsin is shown indicating the positions of the nine 15N-labelled methionine residues, present in each sample Three 13C-labelled residues, [13C1]Ile117, [13C1]Phe54 and [13C1]Phe208 that form spin pairs with labelled methionine residues 118, 56 and 209, present in two separate samples prepared for REDOR experiments are also shown RASMOL was used with coordinates 1c3w [14] from the Protein Data Bank 2153 N- and O-states of BR seen by solid-state NMR N- and O-like states Methionines are found at residues 20, 32, 56, 60, 68, 118, 145, 163 and 209, with only 68 and 163 located in loops and all others located in helices A, B, D, E and G Application of this labelling scheme in combination with cross polarization and magic angle sample spinning (CPMAS) NMR techniques provides well resolved spectra that exhibit chemical shift differences and resonance line broadening in N- and O-like states compared with bR An assignment based on rotational echo double resonance (REDOR) experiments in conjunction with double labelling together with Mn2+-induced quenching and D2O exchange of some of the observed resonances allows a more detailed view of conformational changes and motions within these mutants, which serve as models for the N- and O-like states A J Mason et al A B Results The 15N CPMAS spectrum of [15N]Met purple membrane (Fig 2A) allows the resolution of seven resonances out of nine labelled residues Contribution from the 15N natural abundance (0.37%) from a 26 kDa protein is calculated to be equivalent to 0.9 15 N nuclei per nine labelled residues, and is spread over the full amide spectral region Therefore, it can be considered to be negligible in contrast to 13C labelling Compared to the wild type spectrum and to each other, both N- and O-like preparations of D85N ⁄ T170C (Fig 2B,C) show marked differences Both the N-like and O-like state spectra are characterized by a number of well resolved resonances In general, in the N-like state the resonances appear slightly broader and there is a greater degree of overlap In the O-like state, the number of clearly resolved resonances is reduced with O2 and O4 appearing only as shoulders to the intense O3 resonance A summary of chemical shifts and linewidths resulting from spectral deconvolution is given in Table Resonances for Met20 (bR7) and Met145 (bR6) have been assigned previously in bR using the singlesite mutations M20V and M145H [20,21] (supplementary Fig S1), whilst Met32 (bR2) was tentatively assigned previously as a shoulder resonance using the REDOR technique [21] and confirmed by D2O exchange [20] Here, we sought to assign the remaining resolved methionine resonances by making use of Mn2+-induced line broadening and deuterium exchange of residues located close to the membrane surface [22] in addition to REDOR on [15N]Met ⁄ [13C1]Ile or [15N]Met ⁄ [13C1]Phe membrane preparations REDOR as an assignment technique has been used previously as a selective filter [23] and to assign 2154 C Fig 15N CPMAS spectra obtained for [15N]methionine-labelled (A) purple membranes (bR) in pH buffered H2O, (B) D85N ⁄ T170C membranes at pH 10 (N-like), and (C) D85N ⁄ T170C at pH (O-like) Resonances are labelled from large to small chemical shifts (Table 1) Resonance assignment is discussed in the text Spectra were acquired at 60.82 MHz 15N Larmor frequency, 253 K and kHz sample rotation rate Spectra were deconvoluted using PEAKFIT to obtain the linewidths of overlapping resonances specific proline residues in bacteriorhodopsin [24] Knowing the primary structure of bR allows the generation of unique 15N-13C1 pairs by colabelling FEBS Journal 272 (2005) 2152–2164 ª 2005 FEBS A J Mason et al N- and O-states of BR seen by solid-state NMR Table Summary of chemical shift and full width at half height (FWHH) for all [15N]Met resonances shown in Fig Linewidths were obtained by deconvolution using PEAKFIT Resonance BR (pH 6) N-like (D85N ⁄ T170C, pH 10) O-like (D85N ⁄ T170C, pH 6) Peak and assignment dISO (p.p.m.) FWHH (p.p.m.) bR1 ⁄ Met118 bR2 ⁄ Met32a bR3 ⁄ Met56 bR4 ⁄ Met60b bR5 ⁄ Met209 bR6 ⁄ Met145 bR7 ⁄ Met20 N1c (Met118) 127.8 124.4 123.9 122.5 122.1 120.7 118.0 128.2 127.7 127.3 126.6 123.8 122.9 121.5 120.6 120.0 118.1 127.3 126.8 0.42 0.45 0.53 0.34 0.48 0.84 0.37 0.57 0.65 0.78 0.86 1.24 0.82 0.52 0.72 0.59 0.57 0.56 0.86 124.1 122.9 121.8 120.3 119.9 117.5 2.1 0.71 1.1 0.74 0.45 0.50 N2 N3 ⁄ Met209 N4 ⁄ Met56 N5 ⁄ Met145 N6 ⁄ Met145 N7 ⁄ Met20 O1a O1b (Met118) O2 O3 ⁄ Met209 O4 O5 ⁄ Met145 O6 ⁄ Met20d a The shoulder down field of bR2 is best approximated by a Gaussian with dISO ¼ 125.1 p.p.m., FWHH ¼ 0.822 p.p.m b The shoulder between bR3 and bR4 is best approximated by a Gaussian with dISO ¼ 123.0 p.p.m., FWHH ¼ 0.67ppm c The best deconvolution of N1 has been achieved with at least four Lorentzians d The small peak down field of O6 is best approximated by a Lorentzian with dISO ¼ 118.3 p.p.m., FWHH ¼ 0.71 p.p.m [15N]Met samples with the upstream residue enriched with 13C1 These 15N-13C1 pairs have a strong dipolar coupling which can be used to selectively dephase and therefore assign the related 15N-resonances Applying the REDOR technique to the [15N]Met ⁄ 13 [ C1]Ile bR sample using a short dephasing time of 1.4 ms caused a significant reduction in the intensity of resonance bR1 (Fig 3) Other resonances were unaffected within the limits of the signal-to-noise ratio of the spectrum The observed dephasing was mainly due to the strong dipolar coupling between directly bonded [13C1]Ile117 and [15N]Met118, which are separated by ˚ only 1.3 A (Fig 1) A small additional contribution could also arise from [13C1]Ile119 which, in the 3D crys˚ tal structure of bR (1c3w [14]), is 4.7 A away Therefore, bR1 can be assigned to Met118 in bR The only ˚ other spin pair within a A radius of any [13C1]Ile in FEBS Journal 272 (2005) 2152–2164 ª 2005 FEBS Fig Dephased (S) and nondephased (S0) 15N-detected 13C ⁄ 15N CP REDOR spectra of [15N]Met ⁄ [13C1]Ile purple membranes A significant signal decay of resonance bR1 is observed for a short REDOR dephasing time of 1.4 ms The only directly coupled 15 N–13C spin pair is [15N]Met118 ⁄ [13C1]Ile117, which allows the assignment of bR1 to Met118 Intensity variations of the other signals are mainly due to noise and are discussed in the text Spectra were acquired at 40.52 MHz 15N Larmor frequency, 253 K and kHz sample rotation rate this sample would be [15N]Met56 and [13C1]Ile52, which ˚ are approximately 4.2 A apart This weak dipolar coupling would cause less signal decay at the short dephasing time used here We expect this signal reduction to be below the noise level of this experiment and consider the decay at bR3 as not significant at this stage Further resonances were assigned in [15N]Met ⁄ 13 [ C1]Phe bR, N- and O-like preparations Again using a short dephasing time of 1.4 ms, a significant signal reduction of bR5 was observed (Fig 4A) in bR membranes, while other resonances were unaffected within the noise level of our data In N- and O-like preparations, dephasing was observed for resonances N3 (Fig 4B) and O3 (Fig 4C), respectively The observed dephasing in each case was due to directly bonded [13C1]Phe208-[15N]Met209 Therefore, bR5, N3 and O3 were assigned to Met209 in bR, N-like and O-like states, respectively The N-like preparation was suspended in buffer at pH 10 containing 40 lm Mn2+ to remove the signal from surface-exposed residues (N2, see below) and allow a clearer observation of the dephasing effect on N3 Some additional signal reduction of N4 in the N-like state may be attributed to dephasing of [15N]Met56 by [13C1]Phe54 Extending the REDOR dephasing time from 1.4 to 16 ms causes signal decay for a further resonance, bR3 2155 N- and O-states of BR seen by solid-state NMR A J Mason et al Fig 15N CPMAS and 15N-detected 13C ⁄ 15N CP REDOR spectra of [15N]Met ⁄ [13C1]Phe purple membranes suspended in 40 lM Mn2+ pH buffer REDOR dephasing was applied for 16 ms which completely dephases the signal from resonance bR5 but also shows signal decay for bR3 In this sample, only [13C1]Phe208 and ˚ [15N]Met209 are directly coupled but [13C1]Phe54 is within 3.2 A of [15N]Met56 causing a slower dephasing due to a weaker dipolar coupling Therefore bR3 is assigned to Met56 Spectra were acquired at 40.52 MHz 15N Larmor frequency, 253 K and kHz sample rotation rate Fig Dephased (S) and nondephased (S0) 15N-detected 13C ⁄ 15N CP REDOR spectra of [15N]Met ⁄ [13C1]Phe (A) purple membranes and D85N ⁄ T170C membranes, at (B) pH 10 (N-like) in presence of 40 lM Mn2+, and (C) pH (O-like) Resonances bR5, N3 and O3 show strong decays at 1.4 ms REDOR dephasing time The only directly coupled 15N-13C spin pair is [15N]Met209 ⁄ [13C1]Phe208 which allows assignment of bR5, N3 and O3 to Met209 Spectra were acquired at 40.52 MHz 15N Larmor frequency, 253 K and kHz sample rotation rate 2156 (Fig 5) In the 1c3w 3D crystal structure of bR, ˚ [13C1]Phe54 is located approximately 3.2 A from 15 15 ˚ of any [ N]Met56 No other N labels are within A [13C1]Phe except [15N]Met209 Therefore, bR3 is assigned to Met56 The sample was suspended in buffer containing 40 lm Mn2+ to remove the signal from surface-exposed residues (bR4, see below) and allow a clearer observation of the dephasing of bR3 and bR5 For technical reasons, all REDOR experiments presented here were performed at 40.54 MHz 15N Larmor frequency and at a kHz sample rotation rate (Figs 3–6), compared to 60.82 MHz and kHz for the cross polarization spectra presented in Fig and discussed earlier Therefore, a poorer resolution was achieved and bR2 was not clearly resolved under these conditions In addition, the different line shapes and peak intensities obtained by cross polarization and REDOR are caused by different spin relaxation due to the long delays between rf pulses in the REDOR experiment Mn2+-induced paramagnetic line broadening of NMR signals has been described previously in FEBS Journal 272 (2005) 2152–2164 ª 2005 FEBS A J Mason et al Fig Comparison of 15N CPMAS spectra in the absence or presence of 40 lM Mn2+ (A) [15N]Met purple (bR) membranes at pH with (dotted line) and without (solid line) the addition of 40 lM Mn2+ Resonance bR4 is most affected, which must arise from a residue close enough to the membrane surface to be broadened in the presence of Mn2+ ions Spectra were acquired at 40.52 MHz 15 N Larmor frequency, 253 K and kHz sample rotation rate (B) [15N]Met D85N ⁄ T170C membranes in water buffered at pH 10 (solid line, top) and 40 lM Mn2+ solution also buffered at pH 10 (dotted line, top) The reduction in intensity is due to the broadening of signals resulting from surface-accessible residues Spectra were acquired at 60.82 MHz 15N Larmor frequency, 253 K and kHz sample rotation rate bacteriorhodopsin [22], where it was used to assign residues close to the membrane surface Strong dipole– dipole interactions induce accelerated spin relaxation and a concomitant line broadening in excess of 100 Hz, such that NMR signals from residues close to the membrane surface are suppressed in the CPMAS spectra Due to their location close to the membrane surface, signals from Met32, 60, 68 and 163 are expected to FEBS Journal 272 (2005) 2152–2164 ª 2005 FEBS N- and O-states of BR seen by solid-state NMR broaden upon the addition of Mn2+ In the bR-state a significant reduction of intensity is observed for bR4 (Fig 6A) However, previous D2O exchange experiments [20] which remove signals from exchangeable residues Met32, 68 and 163 [25] did not show an effect on bR4, which indicates that bR4 is Met60 (Fig 6A) In the N-like state (Fig 6B) a significant reduction of intensity was only seen in the region around N2 15 N CPMAS spectra acquired after the incubation of [15N]Met membranes in D2O reveal solvent-exposed residues due to a reduction in cross polarization by exchanging the amide proton with a deuteron In the N-like state, N2 is effectively removed by deuterium exchange (Fig 7A) as is O2 in the O-like spectrum (Fig 7B) The observed effect on resonance N2 is consistent with the detected quenching in the presence of Mn2+ discussed earlier Therefore this signal must arise from a solvent-accessible residue close to the membrane surface (Met32, 68, 163) Differences between N-like spectra affected by Mn2+ quenching (Fig 6B) and deuterium exchange (Fig 7A) could be caused by Met60, but have not been observed This would suggest that the Met60 resonance is of low intensity and ⁄ or largely obscured by the intense resonance assigned to Met209 in the N-like state Other resonances are unaffected by deuterium exchange with the exception of O6 In the N-like state spectrum, resonance N7 occurs at the same chemical shift and with similar intensity as bR7 (Met20), but is slightly broader (Table 1) The intensity of N7 is related to that of resonance O6, which is shifted by only )0.5 p.p.m When the membranes are suspended in D2O in an O-like state, O6 splits into two resonances O6a and O6b (Fig 7B) The additional resonance O6a occurs at 118 p.p.m as bR7 and N7, while O6b has the same chemical shift as O6 The appearance of resonance O6a appears to cause a signal reduction of O6 An explanation would be a change in equilibrium between the N and O-like states caused by resuspending the samples in D2O with a subsequent change in pH This is supported by the detection of a blue shift of kmax by nm in the absorption spectra of O-like samples in D2O (supplementary Fig S2) compared to preparation in H2O N-state samples are shifted by only nm These observations provide further evidence that N7 and O6 correspond to the same residue Discussion The 15N CPMAS spectrum of [15N]Met purple membranes (Fig 2A) allows the resolution of seven resonances, which correspond to the seven methionine 2157 N- and O-states of BR seen by solid-state NMR A J Mason et al Fig Comparison of 15N CPMAS spectra of [15N]Met D85N ⁄ T170C membranes in water and D2O (A) Buffered at pH 10 (N-like state), (B) buffered at pH (O-like state) The spectral subtractions reveal the intensity of the resonances resulting from exchangeable residues (N2, O2) Spectra were acquired at 60.82 MHz 15N Larmor frequency, 253 K and kHz sample rotation rate resonances located in transmembrane helices A, B, D, E and G (bR1–bR7 represent Met118, 32, 56, 60, 209, 145 and 20) The linewidth of the resolved resonances ranges from 0.37 to 2.1 p.p.m The obtained spectral resolution was better than in previously published work [20], which is probably due to the use of a higher magnetic field and faster sample spinning Spectra in Fig were deconvoluted with the minimum number of Gaussian or Lorentzian peaks required to minimize v2 Possible error sources are limited signal-to-noise (1 : 20–1 : 40 from 3–4 mg sample) as well as a small amount of potential isotope scrambling which might account for some background signal Here, no direct contributions from loop resonances Met68 and Met163 were detected However, deconvolution of the spectrum in Fig 2A hints at additional signal contributions to the shoulders seen downfield of bR2 (Met32) and bR4 (Met60) The reduced intensity and line broadening of these loop residues has been proposed to be due to fluctuating motions that interfere with the line narrowing processes of MAS or heteronuclear 1H decoupling during acquisition [22] The reduced spectral intensities of [15N]Met68 and 163 have been also confirmed by deuterium exchange experiments [20] Spectra of the N-like (Fig 2B) and O-like (Fig 2C) states show remarkable differences in line shape and chemical shift when compared to the ground state (Fig 2A) Before discussing the potential meaning of those changes, we need to assess whether they arise from M-, N- and O-state equilibriums or from clean 2158 intermediates We have chosen the D85N ⁄ T170C double mutant, because N- and O-like states can be populated by controlling the pH while the M-state is much reduced compared to the well characterized D85N bacteriorhodopsin mutant At pH 6, D85N contains  95% O-like state and at pH 10 5% O, 20% N and 75% M [18] By introducing an additional T170C mutation, the pKa of M accumulation is raised as shown in Fig 8B The reason is that the M–N transition is coupled to deprotonation of D96 and protonation of the Schiff base For example, in the 3D structure of the N-state [7] T170 faces the cytoplasmic channel at the level of D96 Therefore, a cysteine substitution would alter the hydrophobic pocket and the pKa of D96 and so shift the M–N transition towards N By comparing the singular value decomposition (SVD) analysis performed on D85N [18] with our data (Fig 8B), we estimate the contribution of M-state to our sample at pH 10 to be not more than 30% Opposite to M–N, the N–O transition is coupled to the protonation of D96 and to deprotonation of groups at the cytoplasmic surface Therefore, at low pH, D96 will be protonated and we obtain a sample mainly in O-state as shown for D85N (no M- and very little N-contribution) The O-state shows a characteristic absorbance found at 604 nm Raising the pH here also increases contributions from M and N It is known that the N- and O-states have different absorption maxima at kmax 604 nm and 586 nm, respectively (Fig 8A) The N-state extinction coefficient is lower FEBS Journal 272 (2005) 2152–2164 ª 2005 FEBS A J Mason et al Fig UV ⁄ vis spectra obtained for D85N ⁄ T170C membranes purified by sucrose density gradient centrifugation at 38% (w ⁄ w) sucrose at different pH values (A) The N-like state contains some M-state contribution, which is, however, much reduced compared to D85N (B) and can be estimated to  30% based on the SVD analysis for D85N D85N analysis results and data were taken from [18] (70%) than in the O-state Raising the pH from to 10 accumulates a small M-state population but mainly N-state, which is first seen as a signal reduction of the O-state resonance The question is now to what extent both the N- and O-states are mixed at the experimental conditions we have chosen for our NMR experiments In addition to the fact that the absorption maxima in Fig 8A are clearly separated for the N- and O-state, our 15N CPMAS spectra in Fig also show that both states are not significantly mixed The sharp resonance from Met20 (N7 & O6) is in both cases very well resolved and appears at 118.1 p.p.m (N) and 117.5 p.p.m (O) Both lines are only 0.5 p.p.m wide and would be present simultaneously if samples contained a significant N ⁄ O mixture, which is not the case There is no contribution from O in the N-like sample (pH 10) The only hint for an N-state contribution at pH (O-like state) is a small resonance at 118.3 p.p.m Our deuterium exchange data (Fig 7B) have shown that a resonance at 118.0 p.p.m occurs FEBS Journal 272 (2005) 2152–2164 ª 2005 FEBS N- and O-states of BR seen by solid-state NMR when the N–O equilibrium is shifted towards N We cannot exclude at this point that the resonance at 118.3ppm also arises from N in which case its contribution is estimated to  15% based on the fitted peak size Concluding, we can comment that our samples at pH are mainly found in a clean O-state with only a small potential contribution of 15% N and no M-state At pH 10 we find an approximately 70 : 30 N-like ⁄ M-like mixture but no O-like state Therefore, our spectra are dominated by N- or O-like states, which allows us to discuss the nature of the changes in chemical shift and line shape for each individual resonance in more detail The resonance bR7 assigned to Met20 in bR [20] is located upfield and well separated from the other peaks (Fig 2A) In the N-like state a resonance N7 appears with identical chemical shift but slightly broadened by 0.2 p.p.m and separated from all other resonances by p.p.m Both bR7 and N7 are unaffected by Mn2+-induced line broadening and D2O exchange Therefore it seems reasonable to assume that resonance N7 is also caused by [15N]Met20 In the O-like state, resonance O6 appears 0.5 p.p.m upfield of bR7 and N7 and is separated by 2.4 p.p.m from other residues Deuterium exchange suggests, as discussed in the results section, that O6 and N7 belong to the same residue, probably Met20 This would mean that Met20 has the same chemical shift in bR- and N-like states and changes only by 0.5 p.p.m in the O-like state Therefore it is likely that it occupies the same conformation in bR- and N-like states but may experience a subtle change in conformation or an alteration in local hydrogen bonding on conversion to the O-like state Further up helix A, Met32 is assigned to bR2 [21] in bR The fate of this residue on conversion to the N-like state is uncertain as a peak N2 appearing at a similar chemical shift is of a much greater intensity The intensity of resonance N2 is reduced by adding Mn2+ (Fig 6B) and by deuterium exchange (Fig 7A) which points towards a solvent-accessible residue close to the membrane surface such as Met32, 68 or 163 Met32 is the only helical resonance that is exchangeable [20,25] and as discussed earlier, Met68 and 163 are difficult to detect in the [15N]Met spectrum of bR A stronger contribution in the N-like spectrum would only be expected if either loops EF or BC show much reduced molecular motions, which interfere less with the NMR experiments However, this is currently unknown and we cannot safely discriminate between Met32, 68 and 163 Resonance O2 in the O-like state occurs at the same chemical shift as N2 but appears broader with reduced intensity As for N2, deuterium exchange indicates contributions from residues Met32, 2159 N- and O-states of BR seen by solid-state NMR 68 or 163 (Fig 7B) Whether the observed line broadening is of homogeneous or nonhomogeneous nature i.e caused by altered molecular motions on the NMR time scale or by conformational heterogeneity compared to the bR state, cannot be concluded from these data Met56, located on helix B, gives rise to resonance bR3, as shown by the REDOR experiments discussed earlier A similar assignment in the N-like and O-like states was not possible, as experiments with longer dephasing times were hampered by poor sensitivity However, our data can be used to limit the number of possibilities In the N-like state, resonance N4 is not affected by Mn2+ quenching (Fig 6B) nor deuterium exchange (Fig 7A) Therefore, Met32, 60, 68 and 163 can be ruled out, assuming that solvent accessibility and location close to the membrane surface does not change compared to our wild type samples Furthermore, Met20 and Met209 have been already assigned to N7 and N3, which leaves us with Met56, 118 and 145 In bR Met118 had been assigned to resonance bR1 which is p.p.m downfield of N4 It is therefore considered unlikely that N4 is caused by Met118 However, only a 0.8 p.p.m downfield shift for Met145 or 2.4 p.p.m upfield shift for Met56 would be required Further down helix B and close to the extracellular surface is Met60 which has been assigned to bR4 A clear resonance cannot be assigned to Met60 in the N-like state but Mn2+ induced line broadening shows that it is-likely concealed under the intensity N3 assigned to Met209 In the O-like state, the number of resonances is reduced They appear at different chemical shifts and are generally broadened compared to bR Especially spectral components O2 and O4 underlying O3 appear as broad shoulder resonances The intensities around O2 must belong to deuterium exchangeable residues (Fig 7B) while O3 has been assigned to Met209 The small but broad shoulder resonance O4 (Figs 2C and 7B) could correspond to residual intensity due to Met56 and ⁄ or Met60 The observed broadening of lines could be of homogeneous or nonhomogeneous nature Interestingly, recent research suggests that the cytoplasmic half of helix B, where both Met56 and Met60 are located, adopts motional fluctuations after deprotonation of the Schiff base [26] As discussed previously for loop resonances Met63 and Met163 these fluctuations might cause interference with proton decoupling or magic angle sample spinning [22] Whilst the presence of a signal (N4, N5 or N6) that can be tentatively assigned to Met56 in the N-like state suggests that the fluctuating motions proposed in the cytoplasmic half of helix B above Pro50 not affect the whole helix in the N-state, the absence of an intense signal from Met56 2160 A J Mason et al or Met60 in the O-like state suggests that such fluctuating motions are propagated down helix B as far as Met56 or Met60 in this later stage of the photocycle In the bR state Met118 is the most downfield and intense of the methionine resonances Met118 is observed as a sharp peak bR1 In the N-like state a broad resonance N1 with the same chemical shift as bR1 occurs Deconvolution of N1 indicates at least four identifiable resonances (Table 1) Because of similar chemical shifts relative to bR1, their separation by over p.p.m from the other resolved resonances and the fact that bR1, N1 and O1ab are also unaffected by D2O exchange, we assume that these resonances are also due to Met118 The additional resonances are of similar intensity and are shifted both upfield and downfield in the N-like state This could indicate structural heterogeneity around this residue in the N-like state in the membrane environment On conversion to the O-like state two resonances O1a and O1b are observed The second of the two methionine residues located close to the retinal binding pocket [27], Met145, gives rise to a comparatively broad resonance bR6 The process of introducing purple membrane samples into rotors and into the magnet before running experiments for many hours at 253 K will accumulate considerable amounts of dark adapted (DA) bR compared with light adapted (LA) bR in our samples Met145 has already been identified as a key residue in the dark adaptation of bR [28] and the relatively large linewidth of this resonance is evidence for Met145 being either in two conformations in DA and LA bR or experiencing two slightly differing electronic environments At pH 10, mimicking the N-state, two resolvable resonances N5 and N6 are observed which are close to the bR resonance of Met145 (bR6) The chemical shift of N5 is almost identical to bR6 and N6 is slightly shifted upfield by 0.6 p.p.m In the O-state, resonance O5 appears at the same chemical shift as N6 with a downfield shoulder resonance The small difference in chemical shift and the fact that N5, N6 and O5 are also unaffected by Mn2+ induced line broadening and D2O exchange suggest that N5, N6 and O5 are due to the same residue, Met145 The final methionine residue is Met209, located on helix G The intense resonances N3 and O3 in the N-like and O-like preparations (Fig 2B,C) were assigned to Met209 using the REDOR technique described above The resonance ascribed to Met209 shifts downfield by 0.8 p.p.m compared to the ground-state and becomes the most intense resonance The change in intensity could be due to an increase in cross polarization (CP) efficiency which, in combination with the chemical shift change, would indicate a change in FEBS Journal 272 (2005) 2152–2164 ª 2005 FEBS A J Mason et al conformation and dynamic that is maintained in both N- and O-like states The exact nature of the alteration in dynamic and how it is linked to the small observed changes of orientation of helix G [7,13] is unclear, however, it is possible that an increase in intensity of this resonance could be related to a reduced dynamic in this region as the helix moves from its ground state orientation It is interesting to note that in O and also in N, even when considering small M-state contributions, that some resonances are broadened while others remain sharp Resonances from residues Met118 and Met145 are split into a number of differing lines in contrast with others such as Met20, which remain single peaks Interestingly, these line-broadend residues are located around the retinal indicating heterogeneity in this region Previous solid-state NMR studies of D85N bacteriorhodopsin [29] and Raman studies of D85N ⁄ F42C [30] showed that in the O-like state at pH 6, at least two different retinal conformations are present: 13-cis, 15-syn; and all-trans, 15-anti Despite earlier reports of a completely all-trans chromophore at pH 10.8 [31], it was suggested by solid-state NMR [29] and other studies [32] that a mixture of 13-cis, 15anti and all-trans chromophore, with a predomination of the 13-cis, 15-anti form in a bent binding pocket, exists At pH 10, the residual M-state adds contributions from deprotonated 13-cis, 15-anti retinal to the line broadening The retinal structural heterogeneity is reflected in the chemical shift changes and line broadening that takes place for resonances assigned to Met118 and Met145, which are located close to the retinal binding pocket Other resonances such as Met20 have much smaller linewidths indicating that the protein structure around those labels is rather more homogenous It can be seen from the N- and O-like spectra (Fig 2B,C) that Met118 is strongly affected by the presence of a structurally heterogeneous chromophore Based on the observed linewidths and line shapes, Met118 seems to be more heterogeneous in the N-like spectrum compared with the O- or bR-state spectra If the assignment of N5, N6 and O5 to Met145 is correct, then our data implies that this residue adopts two conformations at pH 10, one similar to the bR state and one that will persist into the O-like state At pH the structural heterogeneity around Met118 would be reduced whilst Met145 would be able to adopt a single conformation Conclusions 15 N CPMAS combined with selective [15N]Met labelling has provided some observations on the FEBS Journal 272 (2005) 2152–2164 ª 2005 FEBS N- and O-states of BR seen by solid-state NMR conformational changes that a number of reporter residues in the transmembrane helices undergo The large chemical shift dispersion amongst the 15N-labelled methionine residues allows almost complete resolution in the bR-state and many interesting spectral features to be identified in mutant membranes mimicking the N and O photointermediates The resolution is sufficient to accurately assign some of the residues The observed conformational heterogeneity and the spectral characteristics, as observed previously in 13 CO-labelled preparations [22], identifies amino acids in helix B that undergo fluctuating motions in the last stage of the photocycle before the protein returns to the bR state While the double mutant used for our study allows a decent separation of states, the D85N ⁄ F42C mutant might be even better suited for future studies A lowered pKa for Asp96 [30] may well have even less heterogeneity at pH 10 and could provide an attractive system for further study using this technique, making for an interesting comparison Recently we have shown that orientational constraints can be determined, in a site-directed manner, by specifically labelling bacteriorhodopsin within the purple membrane with 15N-enriched amino acids in combination with magic angle oriented sample spinning (MAOSS) [33] solid-state NMR methods [20,34] This will allow the observed chemical shift changes resolved in this study to be correlated with a more extensive study of the helix reorientations during the reprotonation phase of the photocycle within the natural membrane, the results of which will be reported elsewhere Experimental procedures Sample preparation Halobacterium salinarum (S9 or L33 expressing D85N ⁄ T170C [19]) were cultured in a synthetic medium (1 L) containing all nutrients requisite for normal growth [35] [15N]l-methionine (0.19 gỈL)1) was added to the medium in place of the usual unlabelled l-methionine After five days incubation in the dark (225 r.p.m., 37 °C), when D660 measurements peaked, the cells were harvested and the purple membrane purified following published procedures [36] Sucrose density centrifugation was performed using a stepped sucrose gradient of 10 mL of each of 45%, 35% and 25% sucrose (w ⁄ w) with centrifugation overnight (83 000 g, Beckmann SW28 rotor, 15 °C) Samples containing purified purple membrane were washed and resuspended in mm Na3 citrate, mm KCl buffer (pH 6) Samples containing protein carrying the D85N ⁄ T170C mutation 2161 N- and O-states of BR seen by solid-state NMR were suspended both in pH buffer as above, to mimic the O-like state or a 10 mm Tris, 15 mm KCl buffer (pH 10) to increase the population of the N-like state For CPMAS experiments, [15N]Met wild type and D85N ⁄ T170C (both pH 10 and pH 6) membranes were prepared However, 13C ⁄ 15N-double labelling was necessary for assignment purposes Therefore [15N]Met ⁄ p13C1]Phe (using 0.13 gỈL)1 [13C1]Phe) wild type and D85N ⁄ T170C membranes and [15N]Met ⁄ [13C1]Ile (using 0.22 gỈL)1 [13C1]Ile) wild type membranes were produced for REDOR experiments [15N]Met wild type and D85N ⁄ T170C membranes at pH 10 were also taken and suspended in buffer containing 40 lm Mn2+, the membranes were then pelleted, frozen and CPMAS spectra acquired immediately Finally, [15N]Met D85N ⁄ T170C membranes were suspended in D2O buffered at apparent pH values of or 10 The membranes were incubated at 30 °C for 48 h before being pelleted as above for CPMAS NMR UV/vis and SDS/PAGE characterization of D85N/T170C membranes During the preparation of D85N ⁄ T170C membranes for NMR studies, two populations of blue membrane could be observed on the sucrose density gradient; a main band at 38% and a diffuse band at 30% sucrose Both bands were collected and analyzed by absorption spectroscopy and SDS ⁄ PAGE (supplementary Fig S2) PAGE analysis reveals a dense band at 26 kDa for all samples; however, the less dense membrane was contaminated with other proteins in agreement with the higher A280 ⁄ A600 ratio seen in the UV ⁄ vis spectra (supplementary Fig S2) Therefore, only membranes collected from 38% sucrose were used for the NMR experiments Absorption spectra were acquired for D85N ⁄ T170C membrane samples suspended in mL of suitable buffer at different pH Wavelength scans from 700 to 260 nm in nm intervals were performed on a Jasco V-550 spectrophotometer (GroßUmstadt, Germany) using a cm light path Figure 8A shows that the chromophore containing protein in the higher density band responds to changes in pH according to the described phenotype for D85N ⁄ T170C bR [19] At pH 10 kmax is found at 586 nm (N-state) and at pH at kmax is 604 nm (O-state) Absorption at 412 nm (M-state) increases with pH Taking into account the contribution of light scattering, usually observed in purple membrane at 412 nm, the M-state contribution is estimated at 30% at this higher pH in the membrane samples used for the NMR experiments This estimation is also supported when compared to the results of singular value decomposition analysis carried out on D85N [18] A direct comparison of the M-state absorption maxima at different pH values in D85N [18] and the system used here also illustrates that the amount of M-state is significantly reduced due to the additional T170C mutation (Fig 8B) 2162 A J Mason et al Solid-state NMR spectroscopy 15 N CPMAS experiments were performed at 60.82 MHz and 40.54 MHz for 15N on Bruker Avance 600 and 400 spectrometers (Karlsruhe, Germany) equipped with mm and mm DVT-MAS probes, respectively A recycle delay of s was used with a contact time of ms, an acquisition time of 49 ms and a spectral width of 50 kHz Optimized 80–100% ramped CP experiments with proton decoupling, using a two pulse phase modulation at 62.5 kHz 1H field were performed at 253 K, at sample rotation rates of kHz and kHz Free induction decays were processed with 16k points, without exponential line broadening prior to Fourier transformation Processed spectra were deconvoluted using peakfit (SeaSolve, Richmond, CA, USA) to obtain the linewidths of overlapping resonances 15 13 N-detected C ⁄ 15N-REDOR experiments were performed at 100.63 MHz ⁄ 40.54 MHz for 13C ⁄ 15N on a Bruker Avance 400 spectrometer equipped with a mm DVT-MAS triple resonance probe, at 253 K and at a sample spinning rate of kHz A standard REDOR pulse sequence according to [37] was used Two equally spaced 13 C p pulses at a field strength of 36 kHz were applied per rotor period A 15N p pulse (40 kHz) in the middle of the dephasing period replaced the 13C pulse and refocused 15 N chemical shifts Dephasing times were varied between and 79 rotor cycles (1.4 and 16 ms) to sample both stronger dipolar couplings arising from directly bonded 15N-13C1 spin pairs as well as weaker dipolar couplings from spin pairs which are separated by more than one bond length Free induction decays were processed with 16k points without exponential line broadening prior to Fourier transformation 15 N chemical shifts were measured relative to an external standard of solid (NH4)2SO4 at 27 p.p.m All NMR experiments were performed on samples containing approximately 3–4 mg of protein Acknowledgements This work was supported by DFG GL307 ⁄ 1–2 The authors thank Leonid Brown for critical reading of the manuscript Single site mutants used previously to assign methionine resonances were provided by Janos Lanyi and Leonid Brown References Stoeckenius W, Lozier RH & Bogomolni RA (1979) Bacteriorhodopsin and the purple membrane of halobacteria Biochim Biophys Acta 505, 215–278 Henderson R, Baldwin JM, Ceska TA, Zemlin F, Beckmann E & Downing KH (1990) Model for the structure FEBS Journal 272 (2005) 2152–2164 ª 2005 FEBS A J Mason et al 10 11 12 13 14 15 of bacteriorhodopsin based on high resolution electron cryo-microscopy J Mol Biol 213, 899–929 Hirai T & Subramaniam S (2003) Structural insights into the mechanism of proton pumping by bacteriorhodopsin FEBS Lett 545, 2–8 Lanyi JK & Schobert B (2004) Local-global conformational coupling in a heptahelical membrane protein: 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fusion peptides J Magn Reson 159, 101–110 Lansing JC, Hu JG, Belenky B, Griffin RG & Herzfeld J (2003) Solid-state NMR investigation of the buried X-proline peptide bonds of bacteriorhodopsin Biochemistry 42, 3586–3593 Seigneuret M & Kainosho M (1993) Localisation of methionine residues in bacteriorhodopsin by carbonyl 13C-NMR with sequence-specific assignments FEBS Lett 327, 7–12 Kira A, Tanio M, Tuzi S & Saito H (2004) Significance of low-frequency local fluctuation motions in the transmembrane B and C a-helices of bacteriorhodopsin, to facilitate efficient proton uptake from the cytoplasmic surface, as revealed by site-directed solid-state 13C NMR Eur Biophys J 33, 580–588 Greenhalgh DA, Farrens DL, Subramaniam S & Khorana HG (1993) Hydrophobic amino acids in the retinal- 2163 N- and O-states of BR seen by solid-state NMR 28 29 30 31 32 33 34 35 36 binding pocket of bacteriorhodopsin J Biol Chem 268, 20305–20311 Ihara K, Amemiya T, Miyashita Y & Mukohata Y (1994) Met-145 is a key 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ordered membrane protein samples under MAS conditions in press Helgerson SL, Siemen SL & Dratz EA (1992) Enrichment of bacteriorhodopsin with isotopically labelled amino acids by biosynthetic incorporation in Halobacterium halobium Can J Microbiol 38, 1181–1185 Oesterhelt D & Stoeckenius W (1974) Isolation of the cell membrane of Halobacterium halobium and its frac- 2164 A J Mason et al tionation into red and purple membranes Methods Enzymol 31, 667–678 37 Gullion T & Schaefer J (1989) Rotational-echo doubleresonance NMR J Magn Reson 81, 196–200 Supplementary material The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4633/EJB4633sm.htm Fig S1 15N CPMAS spectrum of [15N]methioninelabelled M145H purple membranes suspended in pH buffer The resonance at 120.6 p.p.m (Fig 2A, bR6) is absent and is assigned to Met145 Spectra were acquired at 253 K with a MAS frequency of kHz on a Bruker Avance 600 spectrometer Fig S2 UV ⁄ vis spectra of the D85N ⁄ T170C membranes in D2O showed a blue shift of the kmax of between and nm compared with the corresponding preparations in H2O N-like preparation in D2O (black line) kmax is 582 nm compared with 586 nm in H2O whilst the O-like preparation (grey line) has a kmax at 597 nm in D2O compared with 604 nm in H2O Fig S3 SDS ⁄ PAGE (A) and UV ⁄ vis spectra obtained for D85N ⁄ T170C membranes purified by sucrose density gradient centrifugation at 30% (bA) and 38% (cB) sucrose (w ⁄ w) The gel analysis shows that both bands recovered from the sucrose gradient are enriched with  26 kDa protein but the band recovered at 30% sucrose is contaminated with many other proteins UV ⁄ vis spectra were obtained at pH (dashed line), pH (dotted line) and pH 10 (grey line) FEBS Journal 272 (2005) 2152–2164 ª 2005 FEBS ... site mutants thereof, can be used to isolate the conformational transitions of the reprotonation phase of proton pumping In this study we exploited the pH-dependent transitions of the D85N ⁄ T170C... movements during the transitions between the M-, N-, O- and bR-states As illustrated in the discussion of the O-state models, mutants of Asp85 have been useful in the study of the reprotonation. .. [17] The most notable differences are in the extracellular half of the protein and in the loop regions, particularly the BC, DE and EF loops A slight repackaging of the transmembrane helices in the