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Structural parameters of peptides and proteins in biomembranes can be directly measured by solid state NMR of selectively labeled amino acids. The 19F nucleus is a promising label to overcome the low sensitivity of 2H, 13C or 15N, and to serve as a background-free reporter group in biological compounds. To make the advantages of solid state 19F NMR fully available for structural studies of polypeptides, we have systematically measured the chemical shift anisotropies and relaxation properties of the most relevant aromatic and aliphatic 19F-labeled amino acids. In this first part of two consecutive contributions, six different 19F-substituents on representative aromatic side chains were characterized as polycrystalline powders by static and MAS experiments. The data are also compared with results on the same amino acids incorporated in synthetic peptides. The spectra show a wide variety of lineshapes, from which the principal values of the CSA tensors were extracted. In addition, temperature-dependent T(1) and T(2) relaxation times were determined by 19F NMR in the solid state, and isotropic chemical shifts and scalar couplings were obtained in solution.

Dipolar couplings contain information on internuclear distances as well as orientational constraints. To characterize the structure of the antimicrobial peptide gramicidin S when bound to model membranes, two rigid 4-CF 3 -phenylglycine labels were attached to the cyclic backbone such that they reflect the behavior of the entire peptide. By solid state 19 F NMR we measured the homonuclear dipolar couplings of the two trifluoromethyl-groups in oriented membrane samples. Using the CPMG experiment, both the strong couplings within each CF 3 -group as well as the weak coupling between the two CF 3 -groups could be detected. An intra -CF 3 -group dipolar coupling of 86 Hz and a weak inter -group coupling of 20 Hz were obtained by lineshape simulation of the complex dipolar spectrum. It is thus possible to explore the large distance range provided by 19 F-labels and to resolve weak dipolar couplings even in the presence of strong intra -CF 3 couplings. We applied this approach to distinguish and assign two epimers of the labeled gramicidin S peptide on the basis of their distinct 19 F dipolar coupling patterns.

The homonuclear dipolar coupling between the three equivalent 19 F-spins of a trifluoromethyl group, rotating about its threefold symmetry axis, was studied by multipulse solid-state NMR. A modified CPMG sequence was used first to resolve the dipolar splitting of a powder sample, and then to follow its orientation-dependence in uniaxially aligned samples. Our aim is to employ the CF 3 -group as a highly sensitive reporter to describe the mobility and spacial alignment of 19 F-labeled molecules in biomembranes. As an example, the fluorinated anti-inflammatory drug, flufenamic acid, was embedded as a guest compound in lipid bilayers. Undistorted 19 F dipolar spectra of its CF 3 -group were obtained without 1 H-decoupling, revealing a sharp triplet lineshape. When an oriented membrane sample was tilted in the magnetic field, the change in dipolar splittings confirmed that the guest molecule is motionally averaged about the membrane normal, as expected. A different behavior of flufenamic acid, however, was observed under conditions of low bilayer hydration. From this set of orientation-dependent lineshapes we conclude that the axis of motional averaging becomes aligned perpendicular to the sample normal. It thus appears that flufenamic acid induces a hexagonal phase in the membrane at low hydration. Finally, the dipolar 19 F NMR experiments were extended to frozen samples, where no molecular diffusion occurs besides the fast rotation about the CF 3 -axis. Also under these conditions, the CPMG experiment with composite pulses could successfully resolve the dipolar coupling between the three 19 F-nuclei.

Local macromolecular structure can be determined by solid-state NMR measurements of weak dipolar couplings between selectively labeled groups. The nonperturbing use of 2 H, 13 C, or 15 N in biological systems, however, faces drawbacks in terms of a low sensitivity and a comparatively short distance range relative to 1 H. To extend these limitations, we illustrate the use of 19 F as an alternative NMR probe. The Carr–Purcell–Meiboom–Gill (CPMG) multipulse sequence was adapted here to measure homonuclear dipolar couplings between two fluorine labels in static samples at 470 MHz. Two lipids (4,4-DMPC-F 2 , and a difluorinated sterol), which are arranged in liquid crystalline bilayers, serve as models to assess the scope of the technique. In these 19 F-background-free biological samples, weak couplings down to 100 Hz could be resolved directly from the splitting of the pure dipolar powder lineshape, and 1 H-decoupling was not required. Order parameters were determined for the anisotropic motion of the lipids, consistent with their expected behavior in the membrane. Besides measuring the distance-dependent term of the dipolar coupling in powder samples, we have also used oriented membranes to extract additional angular information from the dipolar anisotropy. The strategy presented here thus has the potential to obtain not only the internuclear distance between two labels, but also their angular orientation in the sample, provided the molecules are aligned as a membrane or a fiber.