O ne a p p ro a ch for o b ta in in g h ig h -r eso lu tio n s tr u c tu ra l an d fu n ctio n a l in fo rm a tio n for b iom em b ran es an d th e ir p ro tein s is b y s ta tic so lid -sta te NMR o f o ri e n te d sy stem s. H ere, a g e n e r a l p ro ced u re to a lig n fu lly fu n c tio n a l b io lo g ica l m em b ra n es co n ta in in g large m em b ran e p ro tein s (Mr >30,000) is d escrib ed . T he m eth od , b a sed on th e iso p o te n tia l sp in -d ry u ltra ce n tr ifu g a tio n tech n iq u e , r e lie s on th e cen trifu g a tio n o f m em b ran e fra g m en ts on to a su p p o rt w ith sim u lta n e ous, or su b seq u en t, p a r tia l e v a p o r a tio n o f th e so lv e n t w h ic h a id s a lig n m en t. atu ral m em b ra n es w ith th e red c e ll a n io n ex ch a n g e tran sp ort p r o te in in ery th ro cy tes, b an d 3, an d th e n ico tin ic a c e ty lc h o lin e receptor. O 1997 Academic Press The elucidation of the structural and functional orga nization of biological membranes by various biophysi cal techniques is one of the important questions being addressed in structural biology. The complexity of the systems, however, their supramolecular structure, and their dynamic properties make direct studies particu larly difficult. Specifically, membrane proteins present extraordinary technical difficulties to the most com monly used methods in structural biology, such as crys tallography (1) and solution NMR spectroscopy (2). In 1 To whom correspondence should be addressed, Fax; 01865/275-234 or -259. E-mail: awatts@bioch.ox.ac.uk. 132recent years solid-state NMR spectroscopy (3, 4) on macroscopically oriented lipid bilayers has rapidly emerged as an alternative approach to elucidate struc tural and functional features of membrane-bound pep tides and proteins. In such aligned systems, lipids and proteins are arranged uniaxially around the mem brane, allowing normal orientation of the molecule backbone relative to the substratum. In combination with isotopic labeling (e.g., 2H, 13C, 15N) this NMR ap proach has successfully been used to determine the complete secondary structure of the M2 channel pep tide and fd coat protein (3), while the orientation of the antibiotic peptide magainin has been resolved in bilayers (5). The complete secondary structure of gram icidin and its dynamic properties in membranes have also been obtained using 2H and 15N NMR (6-9). In addition, the complete structure and orientation of deu terated retinal in bacteriorhodopsin at different states of its photocycle has been resolved (10), The average molecular and orientational order of lipids and peptides in liquid crystalline membranes could also be deter mined unambiguously in this way (11-14).Unfortunately, the generation of structural and ori entational information about membrane proteins has been limited mainly to peptides, since no general methods have been available for aligning native and reconstituted membranes containing larger integral membrane proteins in sufficient quality and quantity without affect...

Several close analogues of the noncovalent H؉ /K ؉ -ATPase inhibitor SCH28080 (2-methyl-3-cyanomethyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine) have been screened for activity and examined in the pharmacological site of action by solid-state NMR spectroscopy. TMPIP, the 1,2,3-trimethyl analogue of SCH28080, and variants of TMPIP containing fluorine in the phenylmethoxy ring exhibited IC 50 values for porcine H ؉ /K ؉ -ATPase inhibition falling in the sub-10 M range. Deuterium NMR spectra of a 2 H-labeled inhibitor titrated into H ؉ /K ؉ -ATPase membranes revealed that 80 -100% of inhibitor was bound to the protein, and K ؉ -competition 2 H NMR experiments confirmed that the inhibitor lay within the active site. The active binding conformation of the pentafluorophenylmethoxy analogue of TMPIP was determined from 13 C-19 F dipolar coupling measurements using the cross-polarization magic angle spinning NMR method, REDOR. It was found that the inhibitor adopts an energetically favorable extended conformation falling between fully planar and partially bowed extremes. These findings allowed a model to be proposed for the binding of this inhibitor to H ؉ /K ؉ -ATPase based on the results of independent site-directed mutagenesis studies. In the model, the partially bowed inhibitor interacts with Phe 126 close to the N-terminal membrane spanning helix M1 and residues in the extracellular loop bridging membrane helices M5 and M6 and is flanked by residues in M4.

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Dynamic and structural information has been obtained for an analogue of acetylcholine while bound to the agonist binding site on the nicotinic acetylcholine receptor (nAcChoR), using wide-line deuterium solid-state NMR. Analysis of the deuterium lineshape obtained at various temperatures from unoriented nAcChoR membranes labeled with deuterated bromoacetylcholine (BAC) showed that the quaternary ammonium group of the ligand is well constrained within the agonist binding site when compared with the dynamics observed in the crystalline solids. This motional restriction would suggest that a high degree of complementarity exists between the quaternary ammonium group of the ligand and the protein within the agonist binding site. nAcChoR membranes were uniaxially oriented by isopotential centrifugation as determined by phosphorous NMR of the membrane phospholipids. Analysis of the deuterium NMR lineshape of these oriented membranes enriched with the nAcChoR labeled with N ؉ (CD3)3-BAC has enabled us to determine that the angle formed between the quaternary ammonium group of the BAC and the membrane normal is 42°in the desensitized form of the receptor. This measurement allows us to orient in part the bound ligand within the proposed receptor binding site.2 H NMR ͉ structure ͉ 31 P NMR T he ligand gated ion channel, the nicotinic acetylcholine receptor (nAcChoR), is a member of the four-transmembrane superfamily of receptors that includes ␥-aminobutyric acid, glycine, and the 5-hydroxytryptamine receptors and is involved in the transmission of information across the neuromuscular junction (1). Structurally the nAcChoR is composed of five glycosylated subunits (␣ 2, ␤,␥, ␦) with a total molecular mass of 280 kDa (1). Electron diffraction studies have been used to resolve the structure of the receptor to 4.6 Å and to define how it is conformationally altered upon the binding of acetylcholine (ACh) to the synaptic surface of the protein (2). However, the resolution of the current structures is insufficient to resolve the bound agonist, hindering a molecular understanding of the structural and dynamic events associated with the ACh binding to the agonist site on the nAcChoR.Solid-state NMR has demonstrated its applicability to the study of various dynamic processes occurring within the protein backbone, side chains, and bound ligands (3-11). Furthermore, deuterium NMR has found widespread application to the study of protein dynamics both in soluble proteins in the solid state and membrane proteins in their native environment (3-11). In this study, we have exploited the selective reactivity of ␣-Cys-192͞193 toward bromoacetylcholine (BAC) (12-14) after the reduction of the nAcChoR to covalently incorporate N ϩ (CD 3 ) 3 -BAC. The incorporation of this agonist analogue containing NMR-sensitive nuclei into the agonist binding site has allowed a detailed study of the structure and dynamics of the ligand within the agonist binding site of the nAcChoR. Using the well-characterized lineshapes of deuterated quaternary ammonium com...