Primary Transfer Step in the Light-Driven Ion Pump Bacteriorhodopsin

Publication Date (Web): February 28, 2018 ... Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement betwe...
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Cite This: J. Am. Chem. Soc. 2018, 140, 4085−4091

Primary Transfer Step in the Light-Driven Ion Pump Bacteriorhodopsin: An Irreversible U‑Turn Revealed by Dynamic Nuclear Polarization-Enhanced Magic Angle Spinning NMR Qing Zhe Ni,† Thach V. Can,† Eugenio Daviso,†,‡,§ Marina Belenky,‡ Robert G. Griffin,*,† and Judith Herzfeld*,‡ †

Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States ‡ Department of Chemistry, Brandeis University, Waltham, Massachusetts 02454, United States S Supporting Information *

ABSTRACT: Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the Mo intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. 15N(SB)−1H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and 13C−13C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, 15N−13C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.

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INTRODUCTION Bacteriorhodopsin (bR), a 26 kDa membrane protein found in the cell membranes of archaea, functions as a light-driven ion pump, generating electrochemical gradients that drive ATP synthesis. The seven transmembrane helices of bR enclose a retinylidene chromophore formed by a Schiff base (SB) between a retinal molecule and Lys216. Due to bR’s relative availability and stability, it has in the past served as an ideal model for other members of the rhodopsin family and for GPCRs more generally. Since its discovery in the 1970s, bR has been intensely studied by various methods including: X-ray crystallography,2,3 EM,4−6 IR,7−9 molecular simulations10−12 and magic angle spinning (MAS) NMR.13−16 Although advances in these techniques have led to many insights over the years, the details of bR’s primary, irreversible proton transfer remain unclear. The transduction of light energy involves a series of conformational (Figure S1) and protonation changes with the resolved intermediates shown in Figure 1a. In the absence of light, bR exists in dark adapted (DA) state, a mixture of bR555 and bR568.1 When irradiated, bR555 converts to the more stable bR568, also known as the light adapted (LA) state. bR568 is the active form of bR. Although two carboxylates are close to the SB in the resting state of bR568 (see Figure 1b), protonation of the SB is stabilized by delocalization of both the positive charge of the retinylidene across its polyene chain and the negative © 2018 American Chemical Society

charge of the counterion across a large hydrogen bonded complex comprising Asp85, Asp212, Arg82 and three water molecules. Stable protonation of the chromophore is important for both the first and last steps of the photocycle: protonation pushes the absorption of the retinylidene into the visible region and lowers the barrier for thermal reisomerization. This means that SB deprotonation and reprotonation are temporally distant from the isomerization steps that reorient the SB. Nevertheless, deprotonation and reprotonation need to occur on opposite sides of the transport pathway (first to Asp85 on the extracellular side and then from Asp96 on the intracellular side).18 Some insight into how this occurs has come from 15N NMR spectra of the SB in cryo-trapped intermediates (such as those shown in Figure S2). The 15N chemical shifts indicate that the SB in K has lost contact with its original counterion13 while the SB in functional L (the product of K that eventually relaxes to M) has gained a counterion stronger than the original.14 For the intermediates with deprotonated SBs, the 15N chemical shifts report that Mo has a hydrogen bond partner that is absent in Mn.15 This information about the SB interactions allows the λmax values (see subscripts in Figure 1a) to be interpreted in terms of Received: January 1, 2018 Published: February 28, 2018 4085

DOI: 10.1021/jacs.8b00022 J. Am. Chem. Soc. 2018, 140, 4085−4091

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Journal of the American Chemical Society

Figure 1. Ion-motive photocycle, active site, and proposals for the primary proton transfer pathway of bR. (a) Subscripts give the wavelength (nm) of maximum visible absorption. The dark adapted (DA) state comprises a mixture of the inactive bR555 (13-trans, 15-syn) and the active bR568 (13trans, 15-anti).1 Illumination converts bR555 to bR568, resulting in the light adapted (LA) state. Photoisomerization of the C13−C14 bond, turning the SB to the extracellular side, occurs in LA→J and thermal reisomerization, reorienting the SB to the cytoplasmic side, occurs in N→LA.17 However, the SB deprotonates in L→Mo, with concomitant protonation of Asp85 on the extracellular side, and reprotonates in Mn→N, with concomitant deprotonation of Asp96 on the cytoplasmic side. (b) PDB 1C3W structure of the active site in the resting state3 with the intracellular side at the top and the extracellular side at the bottom. The SB interacts with a complex counterion comprising Asp85, Asp212, Arg82 with the mediation of three water molecules. Dashed lines show hydrogen bonds inferred from internuclear distances. (c) Three pathways proposed by Bondar et al.10 for bR’s primary proton transfer from the SB to Asp85 shown using the PDB 5H2K structure for L:19 (A) the proton is transferred directly from the SB to Asp85; (B) the proton is relayed to Asp85 via Thr89; (C) the proton is transferred first to Asp212 and then relayed to Asp85 via Wat402.

Figure 2. Overall schematic for trapping bR photocycle intermediates and examining them with dynamic nuclear polarization enhanced NMR at 250 GHz/380 MHz. In chronological order: The bR sample is packed in a 4 mm sapphire rotor and placed in the stator housing of the 5-channel probe. The photocycle is initiated by visible laser radiation delivered in situ and specific photocyle intermediates are accumulated via various wavelength and temperature protocols. The photocycle intermediate is then trapped by cooling to 90 K in the dark. The sensitivity of each of the NMR experiments is enhanced using DNP, which involves continuous microwave radiation traveling from the gyrotron to the MAS NMR probe via a corrugated waveguide.

polyene perturbations, indicating single bond torsion in K and torsion of nominal double bonds in both L and early M (Mo), which is gone in late M (Mn).13,15 This electrostatically driven torsion in the first half of the photocycle provides a plausible means for the isomerized SB to avoid contacts on the intracellular side of the transport channel, but it does not provide a pathway for the SB proton to get to Asp85 on the extracellular side in the L→Mo transition. Bondar et al. have explored this issue with QM/MM calculations starting from different crystal structures and identified three different pathways with comparably low energy barriers. (Figure 1c): (A) the proton is transferred directly from the SB to Asp85; (B) the proton is relayed to Asp85 via Thr89; (C) the proton is transferred first to Asp212 and then relayed to Asp85 via Wat402. These pathways involve highly divergent mechanisms because each requires the chromophore to twist to varying degrees and involves different displacements of

surrounding residues. Therefore, identifying the operative pathway requires new information about the active site in the L and Mo intermediates. Here, we employ dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS) NMR to seek such information by targeting the SB N−H bond and the interactions between residues at the active site. NMR is an exceptional tool in that it provides both chemical information (in common with other spectroscopies) and distance information (in common with diffraction and microscopy). In the case of MAS NMR, there is no requirement for either solubilization or crystallization, making it a powerful tool in structural biology.20−24 The major shortcoming of NMR is its low sensitivity, which becomes especially problematic when experiments are focused on a single atom in a large molecule that is trapped in a mixture of states. DNP MAS NMR transfers the ∼660-fold greater electron polarization from a stable free 4086

DOI: 10.1021/jacs.8b00022 J. Am. Chem. Soc. 2018, 140, 4085−4091

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Figure 3. SB 15N−1H distances and SB 1H chemical shifts. (a) 2xDIPSHIFT measurements show that the protonated SB in L has an elongated NH bond and the deprotonated SB in Mo is accepting a H-bond. (b and c) 15N−1H HETCOR spectra showing a downfield 1H chemical shift for all intermediates expect for in Mo, where the 1H resonance is 3.6 ppm, indicative of an alcohol. Note throughout that the subscripts are the wavelengths of maximum visible absorption in nm, except in the case of L where the subscripts are the 15N chemical shifts of the SB in ppm. The pulse sequences are shown in Figure S3a,b.

reprotonated in N, it has not yet found a counterion. This is consistent with previous interpretations of the 15N chemical shifts in L and N.14,15 Lastly, though deprotonated, the SB 15N is found to be weakly dipolar coupled to a proton in Mo, which confirms earlier evidence of hydrogen bonding from the 15N chemical shift.28 Characterization of the Schiff Base 1H. Because the resolved signals of the different intermediates are readily assigned in the 15N spectra, the 2D 15N−1H correlation spectra provide clear assignments of the corresponding 1H frequencies, as shown in Figure 3b,c and summarized in Table 1. Laser irradiation at different temperatures yields different populations of the photocycle intermediates due to different barriers for forward and backward conversion. For example, when trapping the K intermediate, a portion of bR is always found in the bR568 state because there is a photoequilibrium between K and bR568

radical to protons, from which it can be further transferred to nuclei of interest.25−27 The resulting 75-fold enhancement of the bR signal, corresponded to a ∼5,600-fold savings in acquisition time. This massive gain in sensitivity allows for multidimensional experiments despite the low gyromagnetic ratios of 15N and 13C. The DNP MAS NMR experiments are carried out at 90 K which coincidently also traps the various bR photocycle intermediates in situ.13,14 A schematic of the instrumentation and the process is shown in Figure 2.

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RESULTS AND DISCUSSION Schiff Base N−H Bond Length. Figure 3a shows the results of the double dipolar chemical shift (2xDIPSHIFT) experiment (Figure S3a) employed to measure the dipolar coupling, and therefore the distance, between the 15N of the SB and the nearest 1H. These distances are obtained by fitting the data shown in Figure S4. Because the influence of dynamics is assumed to be negligible at cryogenic temperatures, the bond lengths are inferred directly from the measured dipolar coupling and are compared with the 1.04 Å length of a standard amide bond (marked with a dashed line). In the first half of the photocycle, the bond lengths of the protonated SBs are longer than that of the amide bond, as expected given known counterion interactions. In the DA state, the bond length for bR568 is slightly shorter than for bR555 indicating a somewhat weaker interaction with its counterion. It can be seen that the bond length of bR568/LA is consistent in mixtures with all intermediates. The bond length is especially long in L185 and short in N, corresponding to a counterion interaction that is stronger in L185 than in bR568 and weaker in N than in bR568. The former suggests that the SB in L185 is ready to deprotonate and that, while the SB is

Table 1. 15N and 1H Chemical Shifts, and N−H Distance for the Schiff Base in bR Intermediate bR555 bR568 in DA bR568 in LA K L165 (a failed L) L174 (a failed L) L181 (a failed L) L185 (functional L) Mo (early M) Mn (late M) N 4087

15

N (ppm)

173.5 165.4 165.4 156.5 165.4 174.3 181.2 184.9 318.4 312.0 173.3

± ± ± ± ± ± ± ± ± ± ±

0.7 0.6 0.6 0.7 0.6 1.0 1.2 1.0 0.6 0.7 0.7

1

H (ppm)

Distance (Å)

± ± ± ±

0.4 0.4 0.5 0.3

1.10 ± 0.02 1.08 ± 0.03 1.06 ± 0.02

11.1 ± 0.3 11.8 ± 0.4 3.6 ± 0.3

1.06 ± 0.03 1.16 ± 0.03 1.45 ± 0.02

16.0 ± 0.3

1.01 ± 0.02

13.2 12.2 12.2 13.3

DOI: 10.1021/jacs.8b00022 J. Am. Chem. Soc. 2018, 140, 4085−4091

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Figure 4. Thr89−Asp85 Contact. 13C−13C chemical shift correlation RFDR (Figure S3c) spectra showing contact between Asp85-Cγ and Thr89-Cβ in L and Mo, but no detectable contact in bR568 or N intermediates. The three states of Asp85-Cβγ observed in L (174.4, 173.2, and 172.3 ppm) correspond to functional L, nonfunctional L and residual bR568, respectively. Only the Asp85-Cγ at 174.4 ppm correlates to Thr89-Cβ. The change in protonation states of the SB and Asp85 L→Mo results in an upfield shift of the Thr89-Cβ resonance.

at 90 K. The 1H chemical shift of the residual bR568 is consistent across the intermediates, providing another indication that the spectra can be reliably compared with one another. In the intermediates, the 1H resonances are found well downfield except for that in Mo, where it shifts far upfield. Whereas the downfield shift befits a SB proton, the ∼3.6 ppm signal is characteristic of an alcohol. Because Thr89 is the only alcohol in the vicinity, it is evidently the H-bond donor to the deprotonated SB in Mo. Furthermore, because the twist in the chromophore is not yet relaxed in Mo,15 it is likely that the proton on Thr89 in Mo was on the SB in L185. This suggests that the SB counterion in L185 is a highly polarized Thr89, which could result from a strong interaction with the still deprotonated Asp85 that is just one turn away on helix C. In fact, Thr89 is already H-bonded to Asp85 in the resting state (see Figure 1b) and vibrational spectroscopy has found a change in Thr89 H-bonding already in the K intermediate.29 In addition to making Thr89 a counterion for the SB, a tight interaction with Asp85 in the L intermediate would establish the possibility of a relay corresponding to pathway B in Figure 1c. To assess this possibility, we turn to 13C−13C correlation spectra. Thr89−Asp85 Proximity. Figure 4 shows 13C−13C correlations between the Cγ of aspartate residues and various upfield carbons. In these spectra, we see the characteristic upfield shift of the Cγ of Asp85 (from ∼174 to ∼169 ppm) upon protonation in the L→Mo transition. In Mo, the Cγ of Asp85 is clearly correlated with the Cβ of Thr89. As usual, multiple forms of the L intermediate are typically trapped along with some bR568. Interestingly, this mixture is reflected in the Cβγ crosspeak of Asp85, which shows that at least three species are present (consistent with the L panel of Figure 3b). However, only one of these species shows a correlation between the Cβ of Thr89 and the Cγ of Asp85 analogous to that in Mo. This is L185, previously identified as the functional L, i.e., the precursor for Mo.15 The nonfunctional L’s do not establish this proximity between Asp85 and Thr89. Significantly, the change in the protonation states of the SB and Asp85 in going from L to Mo also affects the chemical shift of the Cβ of Thr89 (68.2 to 64.8 ppm). This is consistent with Thr89 being caught in a hydrogen bonded chain between the SB and Asp85.

Meanwhile, no correlation between the two residues is detected in bR568 and N intermediates. Together, this supports the notion that the alcohol group of Thr89 and carbonyl group of Asp85 interact tightly only immediately prior to and immediately after the proton transfer step (i.e., the L and Mo intermediate, respectively). Distance Measurements Across the Active Site Reveal Helix Movement. In the past, X-ray crystallography and EM of inevitably mixed bR intermediates have offered numerous structures with modest resolution and ambiguous interpretations.2,4,6,30 As shown in Figure 3, the resolution of the Cβγ signals for Asp85 and Asp212 allows distance measurements across the active site in individual intermediates. Using 3D FSREDOR (Figure S5) to measure the distances between the Cγ atoms of aspartic acids and the N atom of the SB, we obtain the results presented in Figure 5. As expected for functional groups one turn apart on helix G, the SB−Asp212 distance remains at ∼4.5 Å throughout the photocycle. In contrast, we find a significant variation in the distance between the SB on helix G and Asp85 on helix C. The decrease from bR568 to L is consistent with previous X-ray diffraction.31 This need for inward helix movement might explain why some L’s (i.e., relaxation products of K) are nonfunctional at low temperature.14 This barrier is presumably less of a problem at ambient temperatures where the pump is known to be highly efficient. The persistence of a short SB−Asp85 distance in Mo is another indication (in addition to the SB−Thr89 H-bond and persistent chromophore torsion) that the active site has not yet had a chance to relax. The similarity of the distance found in N to that found in bR568 indicates that once proton transfer has neutralized the SB and Asp85, the helices are free to relax to their original relationship. The helix relaxation (in addition to unwinding of the chromophore torsion) makes the proton transfer irreversible by breaking the ion transfer pathway.

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CONCLUSIONS N−1H, 13C−13C, and 15N−13C correlation DNP MAS NMR spectroscopy converge in pointing to path B in Figure 1c as the pathway by which the SB of bR releases a proton to Asp85 on the extracellular side of the transport pathway even though 15

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chromophore torsion and helix flexing to establish a pathway for proton transfer back to the extracellular side, but the shift in electrostatics by which that is achieved is now clear.

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EXPERIMENTAL SECTION

Sample Preparation. The bacteriorhodopsin (bR) containing purple patches of the Halobacterium salinarum cell membrane were isolated using the method of Oesterhelt and Stoechenius.32 [U−13C, 15 N]-labeled purple membranes were obtained from cells grown on uniformly 13C,15N-labeled peptone that was obtained by anaerobic acid hydrolysis of Methylophilus methylotrophus cells that had been grown on 13C-labeled methanol and 15N-labeled ammonium sulfate.33 [ζ-15N]lysine-labeled purple membranes were obtained from cells grown in a synthetic medium containing L-[ζ-15N]lysine.34 The purple membranes were washed 3 times with 300 mM guanidine hydrochloride at pH 10.0. The sample was pelleted after each wash by centrifugation for 2 h at ∼43,000 g. The washed [U−13C,15N] pellet was mixed with 5 mM AMUPol35 polarizing agent in d8-glycerol/ D2O/H2O (60/30/10 volume ratio) and centrifuged once more. The washed [ζ-15N]lysine pellet was mixed with 20 mM TOTAPOL36 polarizing agent in d8-glycerol/D2O/H2O (60/30/10 volume ratio) and centrifuged once more. Trapping Photocycle Intermediates. Two types of monochromatic light sources were used to generate bR photocycle intermediates: a diode-pumped solid state laser from Coherent (Santa Clara, CΑ) operating at 532 nm (green) and a 1W krypton laser from Cambridge Laser Laboratory (Fremont, CA) delivering 647 nm (red). Each photocycle intermediate was generated according to previous protocols:37 • LA was generated by irradiating dark-adapted bR with green laser light for 5 h at 278 K. • K was generated by irradiating LA with green laser light for 3 h at 90 K. • L was generated by irradiating LA with red laser light for 4 h at 150 K. • Mo was generated by irradiating LA with green laser light for 3 h at 210 K. • N was generated by irradiating bR in LA state with red laser light for 3 h at 240 K. Once formed, each intermediate was cooled to 90 K in the dark, where it was studied by DNP MAS NMR. As can be seen in the spectra, irradiation at different temperatures yields various mixtures of the photocycle intermediates due to different barriers for forward and backward conversion. For example, the K intermediate is always accompanied by some bR568 because there is a photoequilibrium between K and bR568 at 90 K. DNP-MAS NMR Spectroscopy. All experiments were conducted with a DNP instrument comprising a gyrotron producing microwaves at 250 GHz38,39 and a 380 MHz/9T solid state NMR spectrometer equipped with a 4 mm MAS 5-channel (1H, 13C, 15N, microwaves, light) probe capable of controlled cooling to cryogenic temperatures.40−42 Throughout the experiments, the sample spinning was performed with cold nitrogen gas cooled to 90 K and routed to the probe through vacuum jacketed transfer lines. 15 N−1H DIPSHIFT (Figure 3A). N−H bond lengths were measured using the 2D dipolar-doubled 15N−1H dipolar chemical shift (double-DIPSHIFT) correlation experiment43 with ωr/2π = 5 kHz. The pulse sequence is shown in Figure S3a. 1H homonuclear decoupling (ω1H/2π = 100 kHz) was employed in the MREV-8 sequence,44 which has a theoretical scaling factor of 0.477 for single DIPSHIFT, and 0.954 for the corresponding double-DIPSHIFT experiment. The experimental scaling factor at 90 K was obtained using the amide backbone signal as a reference and was found to be between 0.9 and 1.0 of the coupling constant for a typical NH bond of 1.04 Å.45 Each error bar was estimated from a pair of simulated DIPSHIFT curves corresponding to the upper and lower bounds of the measured distance. Integrated SB intensities were deconvoluted using Origin and then fitted using SpinEvolution.46 The integrated SB

Figure 5. Distance measurements across the active site. Dephasing curves from 3D FS-REDOR experiments are used to obtain distances from the SB nitrogen to the Cϒ of Asp85(red) and Asp212 (blue) in the LA, L, Mo, and N intermediates. Simulated curves (black dotted line) are plotted for distances from 4.0 to 5.5 Å in increments of 0.5 Å. S (and reference S0) curves were acquired with (and without) a Gaussian pulse on 15N with varying mixing times from 0 to 25 ms. The SB-Asp85 distance contracts in the first half of the photocycle and relaxes after the primary proton transfer that neutralizes the opposing charges on the two parties. Meanwhile, the SB-Asp212 distance hardly changes.

photoisomerization has turned the SB toward the intracellular side. The 15N−1H spectra show that the deprotonated SB in Mo is accepting a hydrogen bond from an alcohol. The 13C−13C spectra show similar correlations between the carboxyl carbon of Asp85 and the alcohol carbon of Thr89 in L and Mo intermediate, while the chemical shifts of both carbons change in the transition. These experiments were only possible with the sensitivity enhancement provided by the addition of DNP to the experimental protocol. These results provide a new understanding of the structure and function of the L and Mo intermediates. The previously discerned SB counterion in L16 has now been identified as a polarized Thr89. The previous observation of failed L states14 can now be understood as reflecting the difficulty of organizing the hydrogen bonded SB-Thr89-Asp85 complex required for the proton relay in the transition from L to Mo. We now see that this includes not just the previously identified torsion in the chromophore15 but also polarization of Thr89 between the negative charge of Asp85 and the positive charge of the SB. As this process is electrostatically driven, it will be reversed once proton transfer has neutralized the SB and Asp85, consistent with the absence of torsion in the chromophore in the Mn and N states. The resulting breakdown of the primary proton transfer pathway forces the SB to be reprotonated from the intracellular side of the transport channel. The present results also allow a new appreciation of nature’s design of the active site. In the resting state, the carboxyl group of Asp85 is H-bonded by one oxygen to Wat402 and by the other to Thr89. As long as the SB is directed toward the extracellular side, the negative charge of Asp85 is presumably directed disproportionately toward the SB via Wat402. However, once photoisomerization reorients the SB toward the intracellular side, the negative charge of Asp85 is presumably redirected toward the SB via Thr89. It still requires 4089

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intensities are then plotted as a function of evolution time up to one rotor period. 15 N−1H HETCOR (Figure 3B). Two-dimensional 15N−1H HETCOR spectra were recorded ωr/2π = 5 kHz. In the HETCOR pulse sequence (Figure S3B), the homonuclear dipolar coupling is attenuated by the MREV-8 homonuclear decoupling sequence, and the heteronuclear dipolar couplings are averaged by MAS during the t1 evolution period. Subsequently, cross-polarization transfers the frequency encoded 1H polarization to 15N for observation. For cross-polarization, the contact time was 0.15 ms, except for Mo (0.9 ms), with a ω1H/2π = 50 kHz spin lock field on 1H. 1H homonuclear decoupling during the t1 evolution period used a 100 μs dwell time and 32 increments was employed using MREV-8 sequence with ω1H/2π = 100 kHz. 1 H chemical shifts were obtained by multiplying the acquired values by the MREV-8 experimental scaling factors, which were determined using the amide and arginine crosspeaks (not shown) as internal references set to 9.1 and 8.28 ppm, respectively. The experimental scaling factor ranged from 0.453 to 0.547. 1H decoupling (ω1H/2π = 100 kHz) was used during acquisition. The resulting 15N, 1H chemical shifts are summarized in Table S1 with experimental uncertainties determined by the line widths at half height. 13 C−13C RFDR (Figure 4). The RFDR experiment47 (Figure S3C) is initiated by cross-polarization, followed by a chemical shift evolution period, t1. A pair of π/2 pulses flanking the longitudinal mixing period τmix prepares the spin polarization for exchange and returns it to the transverse plane.48 During the mixing period (τmix ∼ 20 ms), rotor synchronized π pulses were applied to mediate exchange via homonuclear dipole−dipole couplings. The π pulses were phase cycled using the XY-16 scheme to compensate for resonance offsets and pulse imperfections. 1H decoupling (ω1H/2π = 100 kHz) was used during mixing and acquisition. Two-dimensional 13C−13C spectra were recorded at ωr/2π = 6.993 kHz using (128, 896) complex points with dwell times (32, 20) μs. Each FID comprised 384 scans with a 7 s recycle delays. 13 C−15N CP-RFDR-REDOR-FS-REDOR (Figure 5). Measurements of the distance between the Asp-13Cγ and the Schiff base 15N were performed using CP-RFDR-REDOR-frequency selective (FS)REDOR49 at ωr/2π = 6.757 kHz and recycle delay 6.8 s. The pulse sequence is shown in Figure S5. Parameters used for the RFDR period are similar to those above and the durations for the RFDR and REDOR filters are τmix = 1.184 and 1.48 ms, respectively. ω1C/2π = 50 kHz was used to generate the π-pulses on the 13C channel during the RFDR, and ω1N/2π = 30 kHz on the nitrogen channel during the REDOR filter and mixing periods. During FS-REDOR (t2), the 180° selective Gaussian pulse lengths were 0.35 and 2.5 ms for Asp-13Cγ and SB-15Nζ, respectively. S and S0 signals, were obtained with and without the 15N selective pulse, and were recorded in alternating fashion at every increment of the indirect dimension. Experimental data were obtained with 128 and 384 transients for t2 < 15 ms and t2 > 15 ms, respectively. The Asp85 and Asp212 signals were integrated in Sparky50 using the same processing parameters for both S and S0. Values of S/S0 were then plotted vs the FS-REDOR mixing time (t2), from 0 to 25 ms. Experimental S/S0 FSREDOR dephasing curves are plotted for LA, L, Mo, and N intermediates. The experimental data were fit with a Bessel function,51 yielding two parameters including scaling factor and internuclear distance. In all cases, scaling factor was found to be ∼0.9, as typically needed to compensate for experimental imperfections.

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Article

AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Robert G. Griffin: 0000-0003-1589-832X Present Address §

Covaris Inc., 14 Gill Street, Woburn, Massachusetts 01801, United States Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by NIH grants EB-001960, EB002804, EB-002026, and EB001035. We thank Ana Nicoleta Bondar and Robert Silvers for valuable discussions, Sudheer Jawla and Richard J. Temkin for support in maintaining the gyrotron, and Ajay Thakkar, Jeffrey Byrant, Mike Mullins and David Ruben for support in maintaining low temperature NMR spectrometer.

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REFERENCES

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b00022. Retinal configurations, 15N spectra of photocycle intermediates, 15N−1H dipolar couplings and several pulse sequences of the experiments (PDF) 4090

DOI: 10.1021/jacs.8b00022 J. Am. Chem. Soc. 2018, 140, 4085−4091

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DOI: 10.1021/jacs.8b00022 J. Am. Chem. Soc. 2018, 140, 4085−4091