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.
Afonin
Sergii
S
Institute of Biological Interfaces, Forschungszentrum Karlsruhe, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
Mikhailiuk
Pavel K
PK
Komarov
Igor V
IV
Ulrich
Anne S
AS
D023181:Antimicrobial Cationic Peptides / Q000737:chemistry; D001643:Bridged Bicyclo Compounds / Q000737:chemistry*; D005617:Chlorofluorocarbons, Methane / Q000737:chemistry; D005461:Fluorine; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary
D023181:Antimicrobial Cationic Peptides; D001643:Bridged Bicyclo Compounds; D005617:Chlorofluorocarbons, Methane; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010455:Peptides; C045866:peptide-Gly-Leu-amide; D005461:Fluorine; D005998:Glycine; C009554:fluoroform