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Solid-State NMR Structure Determination of a Lipid-Embedded

Heptahelical Membrane Protein

Shenlin Wang,1,2 Rachel A. Munro,1,2 Lichi Shi,1,2† Izuru Kawamura,1† Takashi Okitsu,3 Akimori Wada,3

So-Young Kim,4,5 Kwang-Hwan Jung,4,5 Leonid S. Brown,1,2* Vladimir Ladizhansky1,2*

1Department of Physics, University of Guelph, Guelph, Ontario, Canada. 2Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada. 3Department of Organic Chemistry for Life Science, Kobe Pharmaceutical University, Kobe, Japan.

4Department of Life Science, Sogang University, Seoul, South Korea. 5Institute of Biological Interfaces, Sogang University, Seoul, South Korea.

†Present addresses: Department of Medical Genetics and Microbiology, Medical Science Building, University of Toronto, Toronto, Ontario, Canada (L.S.); Faculty of Engineering, Yokohama National University, Yokohama, Japan (I.K.).

Supplementary Materials

Nature Methods: doi:10.1038/nmeth.2635

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Supplementary Figure 1. Representative two-dimensional planes extracted from the 3D HBR2 spectrum of 1,3-ASR.

(a-e) 2D N-CX planes [F1 - F3] extracted at the carbonyl chemical shifts of 178.9 ppm (a), 175.7 ppm (b), 179.6 ppm (c), 171.6 ppm (d) and 172.2 ppm (e). Short- and medium-range cross peaks (1<|i-j|<5) are shown in blue, and long-range cross peaks (|i-j|>4) are labeled in red.

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Supplementary Figure 2. 2D 13C-13C PDSD spectrum collected on 2-ASR with mixing time of 500 ms.

(a) Carbonyl-aliphatic and retC13-aliphatic correlations. (b) Correlations between aromatic and aliphatic carbons. (c) Aliphatic-aliphatic correlations. Cross peaks between retinal carbons retC13 and retC20, and T80Cγ2 are shown in red in (a) and (c). (d) Expanded region showing cross peaks between aromatic side chain carbons and backbone Cα. Unambiguous inter-helical restraints are labeled. (e) All-trans retinal conformer with carbon atoms numbered. The 13C-labeled carbon atoms are shown in red.

Nature Methods: doi:10.1038/nmeth.2635

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Supplementary Figure 3. 2D CHHC1, 2 spectrum of 1,3-ASR.

(a) 2D CHHC spectrum of 1,3-ASR. (b) Expanded region of backbone Cα-aliphatic side chain correlations. Short- and medium-range cross peaks (1<|i-j|<5) are labeled in blue, and long-range cross peaks (|i-j|>4) are labeled in red.

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Supplementary Figure 4. Sequential plots of dihedral angles predicted using 15N and 13C chemical shifts of ASR by TALOS+ program.

(a-b) Backbone Φ and Ψ angles predicted by TALOS+ as “good” are plotted in panel (a) and (b), respectively. Secondary structure derived from TALOS+ predictions is shown on top.

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Supplementary Figure 5. Monomer structure of ASR.

(a) The lowest energy structure of ASR monomer ensemble. α-helices are shown in light green and labeled. Retinal is shown in yellow. (b) An ensemble of 10 lowest energy structures of the ASR monomer.

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Supplementary Figure 6. Backbone H/D exchange of ASR.

(a) Sequential plots of cross peak intensities detected in the 3D NCACX spectra of the ASR sample prepared in H2O-based buffer (grey), and exchanged in D2O for 24 h in the dark (blue). Sample preparation and experimental procedures were described previously.3 The H/D exchange data were published in part in ref. 3, and expanded here based on the most recently obtained chemical shift assignments.4 The secondary structure of ASR derived from SSNMR data is shown on top. (b) The exchangeable residues are shown in blue on the monomer structure extracted from the ASR trimer structure.

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Supplementary Figure 7. FTIR spectrum of 15N-ASR reconstituted in DMPC/DMPA lipids at a protein:lipid ratio of 2:1 (w/w).

The absorption peaks corresponding to the vibration of lipid esters (at ~1740 cm-1), of protein backbone carbonyls (Amide I at ~1650 cm-1), and of backbone C-N-H stretching/bending (Amide II at ~1520 cm-

1) are labeled. The actual protein/lipid ratio was confirmed using the ratio of intensities of the peak of lipid esters and the Amide I band.5

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Supplementary Table 1. Parameters of NMR experiments

Three-dimensional HBR2 Acquisition length (t3/t2/t1) [ms] 25.0 (CX)/8.0 (CO)/12.0 (N) π/2 pulse (1H/13C/15N) [µs] 2.5/5.0/7.0 CP contact time (1H/15N) [ms] 2 CP contact time (15N/13C) [ms] 6 HBR2 mixing time [ms] 70 recycle delay [s] 1.7 number of scans 32 Spectral width (F3/F2/F1) [ppm] 276 (CX)/58 (CO)/60 (N) 1H SPINAL646 decoupling radio-frequency field strength [kHz] 84 CP radio-frequency field strength (1H/15N) [kHz] 64.0/53.0 CP radio-frequency field strength (15N/13C) [kHz] 30.0/41.0 CW decoupling power during HBR2 mixing [kHz] 45 Two-dimensional 13C-13C PDSD Acquisition length (t2/t1) [ms] 25.0/12.0 π/2 pulse (1H/13C) [µs] 2.5/4.0 CP contact time (1H/13C) [ms] 2 PDSD mixing [ms] 100/250/500 recycle delay [s] 1.7 number of scans 64 Spectral width (F2/F1) [ppm] 460/250 1H SPINAL64 decoupling radio-frequency field strength [kHz] 84 CP radio-frequency field strength (1H/13C) [kHz] 54.3/40.0 Two-dimensional CHHC1, 2 acquisition length (t2/t1) [ms] 25.0/15.2 π/2 pulse (1H/13C) [µs] 2.5/4.0 First CP contact time (1H/13C) [ms] 2 Second and third CP mixing time (1H/13C) [µs] 200 1H-1H mixing time [µs] 250 recycle delay [s] 1.7 number of scans 64 Spectral width (F2/F1) [ppm] 310/270 13C CW decoupling during 1H-1H mixing7 [kHz] 65 1H SPINAL64 decoupling radio-frequency field strength [kHz] 84 CP radio-frequency field strength (1H/13C) [kHz] 54.3/40.0

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Supplementary Table 2. List of long-range unambiguous restraints used in step 1 of structure calculations protocol From PDSD spectra of 1,3-ASR

61 VAL C’ 67 ILE Cγ1 61 VAL C’ 67 ILE Cγ2 77 MET Cε 113 ILE Cγ2 77 MET Cε 114 THR Cα 80 THR Cβ 113 ILE Cγ1 80 THR Cγ2 Ret C13 80 THR Cγ2 Ret C20 91 ALA Cβ 165 TYR Cδ1 91 ALA Cβ 165 TYR Cδ2 115 SER Cβ 131 TRP Cε3 132 TYR Cζ 180 PRO Cβ 171 TYR Cζ 216 LEU Cδ1 179 TYR Cζ Ret C20 183 TRP Cη2 188 SER Cβ 183 TRP Cε3 188 SER Cβ From PDSD spectra of 2-ASR 11 TYR Cγ 51 TYR Cγ 11 TYR Cγ 203 CYS Cα 46 TRP Cγ 78 VAL Cβ 60 LYS Cα 70 TYR Cγ 60 LYS Cα 68 ALA Cα 73 TYR Cγ 116 GLY Cα 73 TYR Cα 116 GLY Cα 79 THR Cβ 210 LYS Cε 80 THR Cβ 113 ILE Cβ 80 THR Cγ2 Ret C13 80 THR Cγ2 Ret C20 91 ALA Cα 165 TYR Cγ 179 TYR Cγ 202 PHE Cγ 185 ILE Cα 191 GLY Cα 185 ILE C’ 191 GLY Cα 185 ILE Cβ 191 GLY Cα 186 GLY Cα 191 GLY Cα 187 PRO Cα 191 GLY Cα From HBR2 spectra 61 VAL C’ 67 ILE Cγ2 76 TRP C’ 113 ILE Cγ2 *Restraints involving retinal are shown in red color. Retinal structure is shown in Supplementary Figure 2e.

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Supplementary Table 3. List of distance restraints obtained on 1,3-ASR sample.

experiment type

mixing time

total picked peaks

total manual assignments*

intra-residue

restraints

sequential restraints

short-range restraints

medium-range

restraints

long-range restraints&

inter-monomer restraints

3D-HBR2 70ms 214 201 123 57 3 18 8 0

CHHC 250µs 287 225 200 2 0 23 14 0 PDSD 100ms 816 743 577 121 2 43 23 0 PDSD 250ms 1119 971 685 179 8 99 56 2 PDSD 500ms 1428 1139 733 223 22 161 90 8

Supplementary Table 4. List of distance restraints obtained on 2-ASR sample.

experiment type

mixing time

total picked peaks

total manual assignments*

intra-residue

restraints

sequential restraints

short-range restraints

medium-range

restraints

long-range restraints&

inter-monomer restraints

PDSD 100ms 548 501 306 153 8 34 17 0 PDSD 250ms 1010 894 387 335 42 130 66 0 PDSD 500ms 1348 1130 406 411 73 240 102 0

*Manual assignments were performed to analyze the cross peaks corresponding to intra-residue, sequential, short-range and medium-range restraints. &Long-range restraints were obtained both by manual assignments and automated assignments as described in the supplementary text.

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Supplementary Table 5. NMR and refinement statistics for ASR trimer structures (number of restraints are given per monomer) NMR distance and dihedral constraints (per monomer) Distance constraints Total restraints 2840 Intra-residue 1293 Inter-residue Sequential (|i – j| = 1) 663 Short-range (|i -j| = 2) 97 Medium-range (|i – j| = 3 or 4) 435 Long-range (|i – j| > 4) 211 Intermolecular 6

Intra-monomer PRE restraints (<15 Å) Inter-monomer PRE restraints (<15 Å) Hydrogen bonds

24 11 100

Total dihedral angle restraints φ 186 ψ 186

Restraints statistics

Violations (mean and s.d.) Distance constraints (Å) 0.074 ± 0.057 Max. distance constraint violation (Å) 0.37 Deviations from idealized geometry Bond lengths (Å) 0.002 ± 0.000 Bond angles (º) 0.441 ± 0.010 Impropers (º) 0.228 ± 0.017

Structural precision Average pairwise r.m.s. deviation (Å)b

Heavy 1.27 ± 0.18 Backbone 0.84 ± 0.21 Average pairwise r.m.s. deviation (Å)c Heavy Backbone

2.39 ± 0.26 1.80 ± 0.28

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Structural quality Ramachandran Plot Statistics d

Residues in most favored region (%) 88.9 sdsd Residues in additional allowed region (%) 10.4 Residues in generously allowed region (%) 0.5 Residues in disallowed region (%) 0.2

WHAT IF Z-Scorese

1st generation packing quality -1.96 2nd generation packing quality -3.64 Ramachandran plot appearance -2.18 χ1/χ2 rotamer normality -1.96 Backbone conformation 1.22

Clashscoref

Molprobity score (Å)f 14.71 2.5

bPairwise r.m.s. deviation was calculated between 10 structures, for residues in α-helices, including residues 7-26, 34-55, 70-91, 100-120, 125-147, 160-184 and 196-221. cPairwise r.m.s. deviation was calculated between 10 structures, for all residues. dEvaluated with the program PROCHECK.8 eEvaluated with the WHAT IF web server9, 10 for residues 5-221. fEvaluated with the Molprobity program.11

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References 1. Wilhelm, M., Feng, H., Tracht, U. & Spiess, H.W. 2D CP/MAS C-13 isotropic chemical shift correlation

established by H-1 spin diffusion. J Magn Reson 134, 255-260 (1998). 2. Lange, A., Luca, S. & Baldus, M. Structural constraints from proton-mediated rare-spin correlation spectroscopy in

rotating solids. J Am Chem Soc 124, 9704-9705 (2002). 3. Shi, L., Kawamura, I., Jung, K.H., Brown, L.S. & Ladizhansky, V. Conformation of a seven-helical

transmembrane photosensor in the lipid environment. Angew Chem Int Ed Engl 50, 1302-1305 (2011). 4. Wang, S. et al. Solid-state NMR (13)C and (15)N resonance assignments of a seven-transmembrane helical protein

Anabaena Sensory Rhodopsin. Biomol NMR Assign in the press, DOI: 10.1007/s12104-12012-19421-y (2012). 5. daCosta, C.J. & Baenziger, J.E. A rapid method for assessing lipid:protein and detergent:protein ratios in

membrane-protein crystallization. Acta crystallographica 59, 77-83 (2003). 6. Fung, B.M., Khitrin, A.K. & Ermolaev, K. An improved broadband decoupling sequence for liquid crystals and

solids. J Magn Reson 142, 97-101 (2000). 7. Aluas, M. et al. CHHC and (1)H-(1)H magnetization exchange: analysis by experimental solid-state NMR and 11-

spin density-matrix simulations. J Magn Reson 199, 173-187 (2009). 8. Laskowski, R.A., Macarthur, M.W., Moss, D.S. & Thornton, J.M. Procheck - a Program to Check the

Stereochemical Quality of Protein Structures. J Appl Crystallogr 26, 283-291 (1993). 9. Vriend, G. WHAT IF: a molecular modeling and drug design program. Journal of molecular graphics 8, 52-56, 29

(1990). 10. Rodriguez, R., Chinea, G., Lopez, N., Pons, T. & Vriend, G. Homology modeling, model and software evaluation:

three related resources. Bioinformatics 14, 523-528 (1998). 11. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta

crystallographica 66, 12-21 (2010).

Nature Methods: doi:10.1038/nmeth.2635