Measurement of dipolar couplings in a uniformly (13)C,(15)N-labeled membrane protein: distances between the Schiff base and aspartic acids in the active site of bacteriorhodopsin.

In recent years, a number of magic-angle spinning (MAS) solidstate nuclear magnetic resonance (SSNMR) methods have been developed1 for 13C and 15N resonance assignments in uniformly 13C,15N-labeled peptides and proteins.2 The 13CO, 13CR, 13Câ, and amide 15N chemical shifts can be used to estimate the backbone torsion angles φ and ψ.3 Additional constraints on the local structure in U-13C,15N-labeled systems can be obtained from measurements of the relative orientations of dipolar tensors.4 Long-range dipolar couplings in peptides and proteins can provide valuable information about tertiary structure. However, the accurate determination of weak dipolar interactions in U-13C,15Nlabeled molecules is complicated by the presence of strong couplings.5,6 This problem can be circumvented by controlled “dilution” of the multiple spin system7 or by the use of spectrally selective dipolar recoupling techniques.8,9 To date, selective recoupling techniques have been applied to accurate measurements of multiple long-range 13C-13C10 and 13C-15N9 distances in small U-13C,15N-labeled peptides. In this Communication we demonstrate the application of frequency-selective REDOR (FSR)9 to the measurement of two 13C-15N dipolar couplings in the active site of light-adapted [U-13C,15N]bacteriorhodopsin (bR) in its native purple membrane. The measured distances are in reasonable agreement with ones reported in recent diffraction structures of light-adapted bR, and the NMR methods described are directly applicable to bR photocycle intermediates, for which highresolution diffraction structures are more difficult to obtain. The experiments presented here are the first example of long-range MAS NMR distance measurements in a U-13C,15N-labeled macromolecule. Bacteriorhodopsin (bR) is a 26 kDa integral membrane protein produced by Halobacterium salinarum. The single polypeptide chain forms a bundle of seven transmembrane helices enveloping a chromophore formed by a Schiff base (SB) between retinal and Lys216. Dark-adapted bR comprises two species: bR555 and bR568, with different retinal conformations. Light adaptation of bR, by irradiation with white light, converts bR555 to bR568, which is the starting point of the proton pumping photocycle (see ref 15 for a review on bR). In the one-dimensional MAS spectrum of bR568, the Schiff base nitrogen resonates ∼135 ppm downfield from the ú-NH3 groups of Lys residues and ∼50 ppm downfield from the amide backbone peak.16 This enables the selective inversion of the SB nitrogen and FSR distance measurements9 to 13C nuclei in the active site. Recent diffraction structures of bR568 (from 1.55 to 2.9 Å resolution)17-19 report distances to the SB nitrogen in the 4.35.0 Å range for Asp85 Cγ and in the 4.0-4.4 Å range for Asp212 Cγ (see Supporting Information). Figure 1 shows a region of a 2D RFDR11 13C-13C chemical shift correlation spectrum of dark-adapted [U-13C,15N]bR displaying Asp and Glu side chain methylene to carboxyl cross-peaks. * Correspondence author. E-mail: [email protected]. † Massachusetts Institute of Technology. § Brandeis University. (1) Griffin, R. G. Nature Struct. Biol. 1998, 5, 508. Dusold, S.; Sebald, A. Annu. Rep. Nucl. Magn. Reson. Spectrosc. 2000, 41, 185. (2) Rienstra, C. M.; Hohwy, M.; Hong, M.; Griffin, R. G. J. Am. Chem. Soc. 2000, 122, 10979. Balbach, J. J.; et al. Biochemistry 2000, 39, 13748. Detken, A.; et al. J. Biomol. NMR 2001, 20, 203. McDermott, A.; et al. J. Biomol. NMR 2000, 16, 209. Pauli, J.; Baldus, M.; van Rossum, B.; de Groot, H.; Oschkinat, H. ChemBiochem. 2001, 2, 272. (3) Spera, S.; Bax, A. J. Am. Chem. Soc. 1991, 113, 5490. Cornilescu, G.; Delaglio, F.; Bax, A. J. Biomol. NMR 1999, 13, 289. (4) Feng, X.; Lee, Y. K.; Sandström, D.; Eden, M.; Maisel, H.; Sebald, A.; Levitt, M. H. Chem. Phys. Lett. 1996, 257, 314. (5) Hodgkinson, P.; Emsley, L. J. Magn. Reson. 1999, 139, 46. (6) Fyfe, C. A.; Lewis, A. R. J. Phys. Chem. B 2000, 104, 48. (7) Reif, B.; Jaroniec, C. P.; Rienstra, C. M.; Hohwy, M.; Griffin, R. G. J. Magn. Reson. 2001, 151, 320. (8) Raleigh, D. P.; Levitt, M. H.; Griffin, R. G. Chem. Phys. Lett. 1988, 146, 71. Takegoshi, K.; Nomura, K.; Terao, T. Chem. Phys. Lett. 1995, 232, 424. (9) Jaroniec, C. P.; Tounge, B. A.; Herzfeld, J.; Griffin, R. G. J. Am. Chem. Soc. 2001, 123, 3507. (10) Nomura, K.; Takegoshi, K.; Terao, T.; Uchida, K.; Kainosho, M. J. Biomol. NMR 2000, 17, 111. (11) Bennett, A. E.; Ok, J. H.; Griffin, R. G.; Vega, S. J. Chem. Phys. 1992, 96, 8624. (12) Gullion, T.; Schaefer, J. J. Magn. Reson. 1989, 81, 196. (13) States, D. J.; Haberkorn, R. A.; Ruben, D. J. J. Magn. Reson. 1982, 48, 286. (14) Wishart, D. S.; Bigam, C. G.; Yao, J.; Abildgaard, F.; Dyson, H. J.; Oldfield, E.; Markley, J. L.; Sykes, B. D. J. Biomol. NMR 1995, 6, 135. (15) Haupts, U.; Tittor, J.; Oesterhelt, D. Annu. ReV. Biophys. Biomol. Struct. 1999, 28, 367. (16) de Groot, H. J. M.; Harbison, G. S.; Herzfeld, J.; Griffin, R. G. Biochemistry 1989, 28, 3346. (17) Essen, L. O.; Siegert, R.; Lehmann, W. D.; Oesterhelt, D. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11673. (18) Belrhali, H.; Nollert, P.; Royant, A.; Menzel, C.; Rosenbusch, J. P.; Landau, E. M.; Pebay-Peyroula, E. Structure 1999, 7, 909. (19) Luecke, H.; Schobert, B.; Richter, H. T.; Cartailler, J.; Lanyi, J. K. J. Mol. Biol. 1999, 291, 899. Figure 1. Two-dimensional RFDR 13C-13C chemical shift correlation spectrum of dark-adapted [U-13C,15N]bR displaying Asp Câ-Cγ and Glu Cγ-Cδ cross-peaks. Spectra were recorded at 11.7 T, 12.5 kHz MAS, and -80 °C. To eliminate Asn Cγ and Gln Cδ signals in t2, the 2D RFDR sequence11 was followed by a REDOR12 13C-15N dipolar filter: CPt1-(π/2)ψ-RFDR-(π/2)-REDOR filter-t2. RFDR and REDOR filter lengths were 0.96 and 1.44 ms, respectively. Hypercomplex data were acquired by shifting phase ψ according to Ruben and co-workers.13 The data were acquired as (16, 512) complex points with dwell times (320, 20) μs. Each FID was 512 scans, with a 4.0-s recycle delay, resulting in a total measurement time of ∼18 h. 13C chemical shifts are indirectly referenced to the methyl 1H resonance of DSS.14 12929 J. Am. Chem. Soc. 2001, 123, 12929-12930