Compatibility of the conformationally rigid CF3-Bpg side chain with the hydrophobic coiled-coil interface.

3-(trifluoromethyl)bicyclopent-[1.1.1]-1-yl glycine (CF3-Bpg) has previously been established as a useful 19F NMR label to analyse the structures of oligomeric membrane-active peptides or transmembrane segments. To systematically examine the effect of side chain volume, conformational rigidity, and hydrophobicity of CF3-Bpg in polypeptide environments the amino acid was incorporated into an established coiled-coil based screening system. A single substitution of either valine (position a16) or leucine (position d19) within the hydrophobic core of the heteromeric coiled coil has practically no effect on its structure. Despite its comparatively high hydrophobicity, however, the stiff and bulky side chain of CF3-Bpg is not so well accommodated by the hydrophobic core as it leads to a more pronounced destabilization than observed for other, more polar fluorinated amino acids which carry more flexible side chains. CF3-Bpg is therefore a useful 19F NMR label, though not for monitoring the stability of such helix-helix interactions.

Solid state 19F NMR parameters of fluorine-labeled amino acids. Part II: aliphatic substituents.

A representative set of amino acids with aliphatic 19F-labels has been characterized here, following up our previous compilation of NMR parameters for single 19F-substituents on aromatic side chains. Their isotropic chemical shifts, chemical shift tensor parameters, intra-molecular 19F dipole-dipole couplings and temperature-dependent T1 and T2 relaxation times were determined by solid state NMR on twelve polycrystalline amino acid samples, and the corresponding isotropic 19F chemical shifts and scalar couplings were obtained in solution. Of particular interest are amino acids carrying a trifluoromethyl-group, because not only the 19F chemical shift but also the intra-CF3 homonuclear dipolar coupling can be used for structural studies of 19F-labeled peptides and proteins. The CF3-groups are further compared with CH2F-, CD2F-, and CD3-groups, using both 19F and 2H NMR to describe their motional behavior and to examine the respective linebroadening effects of the protonated and deuterated neighbors. We have also characterized two unnatural amino acids in which a CF3-label is rigidly connected to the backbone by a phenyl or bicyclopentyl moiety, and which are particularly well suited for structure analysis of membrane-bound polypeptides. The 19F NMR parameters of the polycrystalline amino acids are compared with data from the correspondingly labeled side chains in synthetic peptides.

Evaluating the amino acid CF3-bicyclopentylglycine as a new label for solid-state 19 F-NMR structure analysis of membrane-bound peptides.

The conformation, alignment and dynamic behavior of membrane-bound peptides is readily accessible by solid-state (19)F-NMR spectroscopy, but it has been difficult to incorporate suitable (19)F-labelled amino acids into synthetic peptides. To avoid the drawbacks of previously used labels, we have rationally designed and synthesized a novel amino acid that suits all theoretical and practical requirements for peptide synthesis and subsequent (19)F-NMR structure analysis [Mikhailiuk et. al, Angew. Chem. 2006, 118, 5787-5789]. The enantiomerically pure L-form of 3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine (CF(3)-Bpg) carries a CF(3) group that is rigidly attached to the peptide backbone and does not racemize during peptide synthesis. It could be demonstrated for several different peptides that their biological activity is usually not affected by a single label, nor the conformation, as monitored by circular dichroism. Here, we carry out a more detailed structure analysis to evaluate the potential and reliability of CF(3)-Bpg for solid-state NMR, using the well-known alpha-helical antimicrobial peptide PGLa as a test case. We have collected several orientational constraints from the anisotropic (19)F--(19)F dipolar couplings of CF(3)-Bpg in various positions of PGLa embedded in lipid bilayers. These resulting structural parameters are then compared with those previously determined from 4-CF(3)-phenylglycine and 3,3,3-d(3)-alanine labels on the same peptide. The analysis confirms that CF(3)-Bpg does not perturb the alpha-helical conformation of PGLa. Likewise, the helix alignment is shown to follow the established concentration-dependent pattern in realigning from a surface-bound S-state to an obliquely tilted T-state. Hence, the advantages of CF(3)-Bpg over all previously used (19)F-labeled side chains are evident, as they combine ease of chemical incorporation and peptide purification with high NMR sensitivity and absent background signals, allowing a straightforward analysis of the dipolar splittings with no need for chemical shift referencing without any ambiguity in the sign of the couplings.