NMR MEASUREMENT OF DIPOLAR COUPLINGS IN PROTEINS ALIGNED BY TRANSIENT BINDING TO PURPLE MEMBRANE FRAGMENTS

One-bond internuclear dipolar couplings in molecules that are aligned relative to an external magnetic field constrain the orientations of the corresponding vectors relative to the molecule’s alignment frame.1-3 Such couplings can be measured easily if the molecular alignment is very weak, such that only the large one-bond effects are observed as a change in the corresponding one-bond J coupling, and broadening resulting from more remote interactions is negligible. For biological macromolecules such as proteins and nucleic acids, the required small degree of alignment with the magnetic field sometimes can be obtained as a result of their own magnetic susceptibility anisotropy,2,3 or more generally, it can be induced by dissolving them in an anisotropic medium.1 Lyotropic liquid crystalline media, consisting of planar phospholipid particles known as bicelles,4 are particularly useful for this purpose.5 Very recently, it has been demonstrated that nematic phases of rod-shaped virus particles can also be used for obtaining a tunable degree of solute alignment.6,7 In both cases solute alignment is contingent upon arrangement of individual particles (bicelles or viruses) in a liquid crystalline phase, which collapses below a given threshold concentration. Therefore, there is a lower limit for the degree of solute alignment which can be obtained in such media. Also, there are a number of proteins which destructively interfere with the bicelle liquid crystalline phase and for which dipolar couplings in such media cannot be measured.5,6 Similarly, it is likely that certain systems will prove unsuitable for measurement in the virus-based media. If a macromolecule can be studied in two or more different oriented media, which in general yield different macromolecular alignment tensors, this dramatically increases the structural information contained in the dipolar couplings.8 Here, we demonstrate that a suspension of planar purple membrane (PM) fragments, containing bacteriorhodopsin (BR), can be used to yield the required weak degree of macromolecular alignment in a strong magnetic field. The magnetic susceptibility anisotropy of these PM fragments is dominated by the membrane spanning helices of BR. Their large size (diameter of 0.2 to 2 μm)9 and high BR content (75%) result in essentially full alignment of individual particles at field strengths g10 T.9 Unlike the liquid crystalline case, there is no critical lower threshold for their concentration. The PM fragments are highly negatively charged, and the average alignment of two water-soluble proteins is shown to be dominated by electrostatic interactions. The solute alignment tensor obtained in the PM medium, therefore, is expected to be quite different from that in virusor bicelle-based liquid crystals. Measurement of one-bond dipolar couplings is demonstrated for two 15N-labeled proteins, the VR domain of the human T-cell receptor, which is one of the proteins which could not be studied in the bicelle medium,5 and ubiquitin which has been studied extensively by NMR and crystallography.10,11 Figure 1 shows a small region of the IPAP 1H-15N HSQC spectrum,12 recorded for 0.4 mM of the VR domain, in a suspension containing 3 mg/ mL purple membrane fragments, 100 mM NaCl, pH 7.1. Substantial deviations from the isotropic JNH splitting (ca. 94 Hz) can be seen, indicating significant alignment. In the frame of the diagonalized molecular alignment tensor, the dipolar couplings can be described by the equation