Biomedical subjects

S L Grage

Publications and source records attributed to S L Grage.

3 recordsLinked to original sources

PubMed2001

Membrane-bound structure and alignment of the antimicrobial beta-sheet peptide gramicidin S derived from angular and distance constraints by solid state 19F-NMR.

The antimicrobial properties of the cyclic beta-sheet peptide gramicidin S are attributed to its destabilizing effect on lipid membranes. Here we present the membrane-bound structure and alignment of a derivative of this peptide, based on angular and distance constraints. Solid-state 19F-NMR was used to study a 19F-labelled gramicidin S analogue in dimyristoylphosphatidylcholine bilayers at a lipid:peptide ratio of 80:1 and above. Two equivalent leucine side chains were replaced by the non-natural amino acid 4F-phenylglycine, which serves as a highly sensitive reporter on the structure and dynamics of the peptide backbone. Using a modified CPMG multipulse sequence, the distance between the two 19F-labels was measured from their homonuclear dipolar coupling as 6 A. in good agreement with the known backbone structure of natural gramicidin S in solution. By analyzing the anisotropic chemical shift of the 19F-labels in macroscopically oriented membrane samples, we determined the alignment of the peptide in the bilayer and described its temperature-dependent mobility. In the gel phase, the 19F-labelled gramicidin S is aligned symmetrically with respect to the membrane normal, i.e., with its cyclic beta-sheet backbone lying flat in the plane of the bilayer, which is fully consistent with its amphiphilic character. Upon raising the temperature to the liquid crystalline state, a considerable narrowing of the 19F-NMR chemical shift dispersion is observed, which is attributed the onset of global rotation of the peptide and further wobbling motions. This study demonstrates the potential of the 19F nucleus to describe suitably labelled polypeptides in membranes, requiring only little material and short NMR acquisition times.

Anti-Bacterial Agents↗

PubMed2000

Orientation-dependent (19)F dipolar couplings within a trifluoromethyl group are revealed by static multipulse NMR in the solid state.

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.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1999

Structural parameters from 19F homonuclear dipolar couplings, obtained by multipulse solid-state NMR on static and oriented systems.

Local macromolecular structure can be determined by solid-state NMR measurements of weak dipolar couplings between selectively labeled groups. The nonperturbing use of 2H, 13C, or 15N in biological systems, however, faces drawbacks in terms of a low sensitivity and a comparatively short distance range relative to 1H. To extend these limitations, we illustrate the use of 19F 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-F2, and a difluorinated sterol), which are arranged in liquid crystalline bilayers, serve as models to assess the scope of the technique. In these 19F-background-free biological samples, weak couplings down to 100 Hz could be resolved directly from the splitting of the pure dipolar powder lineshape, and 1H-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.

Feasibility Studies↗