The conformation of bacteriorhodopsin loops in purple membranes resolved by solid-state MAS NMR spectroscopy.

Membrane proteins (and rhodopsin-like G-protein coupled receptors (GPCRs), in particular) are of significant biological and medical importance since they represent over 50% (GPCRs 25%) of current drug targets. However, the structure determination of membrane proteins is challenging: currently they account for < 1% of the unique protein structures deposited in the Protein Databank. X-ray crystallography has been used to make major contributions towards the structure determination of membrane proteins, but it suffers from the fact that the proteins are rarely crystallized in their native lipid environment or are unmodified, and exposed loop regions are often either dynamic and not visible, or involved in crystal contacts. NMR spectroscopic studies of membrane proteins in solution are generally also reliant on an artificial detergent environment and are also limited by protein size. Solid-state NMR (ssNMR) spectroscopy, in contrast, has the advantage that membrane proteins can be studied in a lipid environment. Although ssNMR does not suffer from the same intrinsic size limitation as solution NMR spectroscopy, spectral overlap is often severe for large proteins and hampers their study. However, magicangle-spinning (MAS) NMR spectroscopy, in particular, has been used to make substantial methodological advances in recent years, and the first membrane protein structures have now been determined using this technique. Herein we report on how solid-state MAS NMR spectroscopy can be used to complement X-ray crystallographic studies of a large seven transmembrane (7TM) helical protein by validating and redefining the loop structures. The structure of bacteriorhodopsin (bR) has been determined in a range of two(2D) and three-dimensional (3D) crystalline environments with the loops showing the highest degree of structural variation. Solid-state MAS NMR spectra of uniformly [C,N]-labeled bR in its native purple membrane have been used to assign the signals of the loop regions of the protein. Extraction of dihedral angle information from chemical shifts has allowed us to validate several loop conformations in the crystal structure and recalculate the structure where there are differences in conformation. Ab initio assignment of the resonances of the loop regions of bR was carried out using 2D DARR spectra (mixing times of 15 and 50 ms) and 3D NCACX (20 ms), 3D NCOCX (20 ms), 3D CANCO and 3D CAN(CO)CX (45 ms) spectra. Assignment of the loops is made possible by the fact that the loop resonances are generally well separated and amenable to assignment in contrast to many of the helical regions, where leucine and valine resonances, in particular, exhibit intractable degrees of spectral overlap. Figure 1 shows the assignment of the section Met68–Gly72 in the BC loop as an example; further 2D spectra and strip plots are provided in the Supporting Information (Figures S1–S3). In total, we have assigned roughly 55% of loop residues covering all loops, except for the CD loop, as well as several residues located in the helices (Figure 2, Table S2 in the Supporting Information, and BMRB Accession code 17361). Interestingly, residues from all loops (except those in the unassigned CD loop) are visible in our cross-polarization (CP)-based spectra; this is in contrast to the spectra of sensory rhodopsin II from Natronomonas pharaonis (NpSRII) where most loops were visible only in INEPT-based spectra, reflecting a higher degree of loop mobility in NpSRII. Our observations are more similar to those made in a recent study of proteorhodopsin in which only isolated residues were observed in INEPT-based spectra. A C,C INEPT-COSY spectrum of bR contains resonances with random-coil chemical shifts from amino acid types that are consistent with the Nand C-terminal tails (see Figures S4 and S5 in Supporting Information). Some chemical shifts for side chains in non-random-coil conformations are also found for residues Lys, Glu, Ala, and Ser, which may belong to the KAES motif in the EF loop. Sequential [*] Dr. V. A. Higman, Dr. P. J. Judge, Prof. A. Watts Department of Biochemistry, University of Oxford South Parks Road, Oxford, OX1 3QU (UK) E-mail: anthony.watts@bioch.ox. ac.uk Dr. K. Varga Department of Chemistry, University of Wyoming Laramie, WY 82071 (USA)