Mechanically, magnetically, and "rotationally aligned" membrane proteins in phospholipid bilayers give equivalent angular constraints for NMR structure determination.

The native environment for membrane proteins is the highly asymmetric phospholipid bilayer, and this has a large effect on both their structure and dynamics. Reproducing this environment in samples suitable for spectroscopic and diffraction experiments is a key issue, and flexibility in sample preparation is essential to accommodate the diverse size, shape, and other physical properties of membrane proteins. In most cases, to ensure that the biological activities are maintained, this means reconstituting the proteins in fully hydrated planar phospholipid bilayers. The asymmetric character of protein-containing bilayers means that it is possible to prepare either oriented or unoriented (powder) samples. Here we demonstrate the equivalence of mechanical, magnetic, and what we refer to as "rotational alignment" of membrane proteins in phospholipid bilayer samples for solid-state NMR spectroscopy. The trans-membrane domain of virus protein "u" (Vpu) from human immunodeficiency virus (HIV-1) and the full-length membrane-bound form of fd bacteriophage coat protein in phospholipid bilayers are used as examples. The equivalence of structural constraints from oriented and unoriented (powder) samples of membrane proteins is based on two concepts: (1) their alignment is defined by the direction of the bilayer normal relative to the magnetic field and (2) they undergo rapid rotational diffusion about the same bilayer normal in liquid crystalline membranes. The measurement of angular constraints relative to a common external axis system defined by the bilayer normal for all sites in the protein is an essential element of oriented sample (OS) solid-state NMR.