Journal of Biomolecular NMR, 8 (1996) 1 14 1 ESCOM J-Bio NMR 356 Macromolecular structural elucidation with solid-state NMR-derived orientational constraints R.R. Ketchem, K.-C. Lee*, S. Huo** and T.A. Cross*** Center for Interdisciplinary Magnetic Resonance at the National High Magnetic Field Laboratory, Institute of Molecular Biophysics and Department of Chemistry, Florida State University, Tallahassee, FL 32306, U.S.A. Received 20 December 1995 Accepted 25 March 1996 Keywords." Gramicidin A; Ion channels; 2H NMR; Side-chain conformation; Membrane protein structure; Oriented bilayers Summary The complete structure determination of a polypeptide in a lipid bilayer environment is demonstrated built solely upon orientational constraints derived from solid-state NMR observations. Such constraints are obtained from isotopically labeled samples uniformly aligned with respect to the B 0 field. Each observation constrains the molecular frame with respect to B0 and the bilayer normal, which are ar- ranged to be parallel. These constraints are not only very precise (a few tenths of a degree), but also very accurate. This is clearly demonstrated as the backbone structure is assembled sequentially and the i to i+6 hydrogen bonds in this structure of the gramicidin channel are shown on average to be within 0.5 A of ideal geometry. Similarly, the side chains are assembled independently and in a radial direction from the backbone. The lack of considerable atomic overlap between side chains also demonstrates the accuracy of the constraints. Through this complete structure, solid-state NMR is demonstrated as an approach for determining three-dimensional macromolecular structure. Introduction The characterization of protein structure in anisotropic environments is one of the current frontiers in the field of structural biology. Solid-state NMR spectroscopy (SS- NMR) of such biological systems has been developed in recent years to respond to this challenge. Both distance (Gullion and Schaefer, 1989; Griffiths and Griffin, 1993) and orientational (Cross and Opella, 1983; Shon et al., 1991; Ketchem et al., 1993) constraints have been demon- strated as quantitative structural constraints in protein and polypeptide structures, as recently reviewed for mem- brane-bound systems (Cross, 1994; Cross and Opella, 1994). While numerous examples of these constraints have been reported, no complete structures have heretofore been achieved. The method for analyzing orientational constraints to achieve a complete atomic-resolution back- bone and side-chain description of a polypeptide, gramici- din A, in a hydrated lipid bilayer is presented here. The complete structure of this channel in a lipid environment has not been achieved previously. A new approach is needed to attack protein structures in anisotropic environments. The formation of three-di- mensional crystals of membrane proteins has proven to be exceptionally difficult and consequently few X-ray crystal structures have been achieved for such proteins (Michel and Deisenhofer, 1990; Weiss et al., 1991; Schiffer et al., 1992; Kuhlbrandt et al., 1994). When it has been possible to prepare two-dimensional crystals, these have been used to characterize membrane proteins through electron dif- fraction (Henderson et al., 1990; Wang and Kuhlbrandt, 1991; Jap et al., 1991; Kuhlbrandt et al., 1994). Solution NMR has also had limited success because the detergents, or lyso-lipid molecules that are used to solubilize mem- brane proteins, add considerably to the protein molecular weight and increase the global correlation time. There- fore, solution NMR has been restricted to small mem- brane proteins such as glucagon (Braun et al., 1983), *Present address: Department of Chemistry and Biochemistry, University of Maryland, P.O. Box 223, College Park, MD 20742, U.S.A. **Present address: MB-2, The Scripps Research Institute, 10666 N. Torrey Pines Road, La Jolla, CA 92037, U.S.A. ***To whom correspondence should be addressed. 0925-2738/$ 6.00 + 1.00 9 1996 ESCOM Science Publishers B.V.