Paramagnetic Relaxation Enhancement

Distance measurement is an essential tool for structure determination from NMR spectroscopy. However, the range of distances that can be observed between protons is limited to 5 A under good conditions 7 A. This is due to the coupling power of two interacting spins that is given by the product of the squares of their gyromagnetic ratios in eqn [2]. By replacing one of the spins by an electron spin, the relaxation can be enhanced over the proton-proton relaxation by a factor of 6602 resulting in an extension of the distances to be accessible by a factor of 6601/3 = 8.7. Therefore, distances of 30 to 40 A become accessible. Paramagnetic tagging has been successfully used for the characterization of large proteins32 and membrane proteins investigated in micelles33 as well as partially folded proteins in solution.34

Slice of 3D NOESY-HSQC

2D NOESY

2D NOESY

V56 Hy' V93 Hy' V15 Hy' V68 Hy'

0Q-CHHC

0Q mixing

CH,CH2 only

Figure 8 (a) 2D liquid-state NOESY of a globular protein showing overlap in the aliphatic region (left), and removal of the overlap by recording a 3D experiment (right). (b) 0Q-correlation experiment representing proton-proton distances detected on the carbons bound to the involved protons, respectively.

3.22.2.3 Dipolar Couplings

Dipolar couplings are averaged out in isotropic solution, but can be reintroduced if the isotropic orientation distribution is disturbed. Technically speaking, the anisotropy of the solution must have a rank two component (American football shape) because the dipolar coupling is also a rank two interaction between two nuclei. The anisotropy of the orientation distribution can be visualized by an ellipsoid that is fixed in the molecular frame. The lengths of the principal axes reflect the probability of finding that axis along the magnetic field. Alignment of proteins in water can be achieved by using intrinsic or engineered anisotropic magnetic susceptibilities35 or by dissolving the protein in an anisotropic medium.36 To scale the dipolar couplings to a value that is smaller than the respective J coupling, alignment of the order of 10 - 3 is desired. Except for the scaling, the spectrum of the aligned protein is then identical to the spectrum that this protein would yield in a single crystal that is switched fast between the three main axes of the alignment tensor with the populations according to the lengths of the axes.37 Dipolar couplings are mainly applied to improve the precision of structures, to investigate protein-protein complexes when few NOEs are measured and to investigate dynamics from dipolar couplings. These applications are summarized in recent reviews.38

It is remarkable that the orientation information that is reintroduced by alignment in solution is not easy to recover for powder samples in solid-state NMR. At least in micro- or nanocrystalline samples, texture effects known from powder pattern x-ray analysis could in principle be used to achieve a nonuniform distribution of orientations of the molecules with respect to the magnetic field. However, by MAS spinning the dipolar couplings are averaged out for each molecular orientation individually, which prevents small texture effects from being used in solid-state NMR spectroscopy under MAS spinning.

Alternatively, membrane peptides or proteins can be macroscopically oriented on glass plates or in the magnetic field.39'40 There are two methods for mechanically aligning lipid bilayers39: deposition from organic solvents followed by evaporation and lipid hydration, and fusion of unilamellar reconstituted lipid vesicles with the glass or polymer surface. Maintaining a constant hydration level of the sample is critical. For this reason, stacked glass plates are in general placed in thin polymer films that achieve heat-sealing and hence stable sample hydration. In addition, bicelles have been used to study membrane proteins by NMR.41 They represent molecular aggregates composed of long-chain phospholipids (such as 1,2-dimyristoyl-s«3-glycerophosphocholine) and either short-chain lipids or surfactants. The long-chain lipids are organized into planar bilayers with the short-chain lipids arranged in a rim surrounding the bilayer edges. Bicellar solutions are lyotropic liquid crystalline solutions and can form a nematic phase that aligns in the magnetic field. The orientation of these bicelles has been shown to be affected by the addition of lanthanide ions.

While residual dipolar couplings Dkf reintroduced in solution carry distance information r—3, they also reflect information on the orientation of the respective internuclear vector with respect to the alignment tensor measured with the polar angles 9¡¡ and fk/. Da is the axial component of the alignment tensor while R is its rhombicity. The axial component can be understood as the difference between the length and the diameter of the 'American football' mentioned above. The rhombicity indicates a deviation from the axial symmetry of the football.

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