Solid-state NMR spectroscopy is emerging as a method ing during data collection (due to the demand for high RF power for glass plate experiments using flat coil probes) capable of describing the structures and dynamics of membrane proteins that in the past have been largely inaccessible complicates the maintenance of adequate sample hydration.
The preparation of samples oriented on glass plates is also to the traditional experimental methods of structural biology, X-ray crystallography, and solution NMR spectroscopy (1). time consuming and tedious. In efforts to increase sensitivity, as well as simplify sample preparation, membrane systems There are two general approaches to membrane-protein structure determination by solid-state NMR spectroscopy. In that orient spontaneously in magnetic fields are being developed. one, unoriented or powder samples are examined using magic-angle spinning methods which enable either homo-Membrane systems composed of mixtures of phospholipids and detergents are known to form bilayer discs of rather or heteronuclear dipolar couplings to be used for distance measurements (2). In the other, orientational constraints for uniform size which orient in an applied magnetic field (10).
The addition of detergent breaks up the extended bilayers bonds and chemical groups are derived from spectral parameters observed in proteins embedded in uniaxially oriented into disc-shaped micelles (bicelles) in which detergent molecules stabilize the edge of phospholipid bilayers. These bi-lipid bilayers. In this Communication, we compare spectra of a uniformly 15 N-labeled membrane protein reconstituted celles typically orient with the bilayer normal perpendicular to the applied magnetic field, as illustrated in Fig. 1G, since into phospholipid bilayers that are magnetically oriented parallel and perpendicular to the direction of the applied mag-the diamagnetic susceptibility (Dx) of phospholipids is negative and the principal axis of the tensor lies along the aver-netic field.
There are special spectroscopic properties associated with age direction of the long axis of the acyl chains. (For Dx less than zero, the energetically favorable orientation of the uniaxially oriented samples in which the axis of orientation lies parallel to the direction of the applied magnetic field molecule is with the principal axis perpendicular to the applied magnetic field.) These perpendicular magnetically ori-(3). The spectra of samples oriented parallel to the field are remarkably simple in that they consist of single-line enting systems have been used to describe the structure and dynamics of a series of glycolipids (10, 11). Initial results resonances (or multiplets) rather than powder patterns for each isotopically labeled site, even in the absence of molecu-have also been obtained using perpendicular orienting systems for both surface and transmembrane peptides ( 12). lar motion. The observed resonance frequencies (or splittings) are directly related to the orientations of individual Efforts have been made to change the sign of the net magnetic anisotropy of the bicelles, thereby flipping them atoms or bonds relative to the direction of the applied magnetic field. This is the reason that spectroscopic measure-to where their bilayer normals are parallel to the magnetic field. Attempts to flip the bicelles through the addition of ments on oriented samples provide the basis for protein structure determination (4). aromatic compounds (13) have met with limited success. However, paramagnetic lanthanides with the appropriate In the past, macroscopic uniaxial orientation of membrane bilayers was generally accomplished mechanically by sand-sign of the susceptibility (i.e., Eu /3 , Er /3 , Tm /3 , Yb /3 ) have recently been shown to produce homogeneous, well-wiching phospholipid bilayers between glass plates (5-7). Although very high degrees of orientation have been ordered orienting systems with bilayer normals parallel to the applied field ( 14), as illustrated in Fig. 1H. As the magnetic achieved for some systems, e.g., gramicidin (8) and coat protein (9), the use of glass plates is experimentally de-susceptibility of paramagnetic compounds can be as much as two orders of magnitude larger than diamagnetic suscepti-manding. Low sensitivity results from filling the majority of the coil volume with glass, rather than sample. Sample heat-bilities, only very small amounts (Ln 3/ /DMPC molar ratios 91