19F labeling

Here, we discuss a solid-state 19F-NMR approach that has been developed for structural studies of MAPs in lipid bilayers, and how this can be translated to measurements in native biomembranes. We review the essentials of the methodology and discuss key objectives in the practice of 19F-labelling of peptides. Furthermore, the preparation of macroscopically oriented biomembranes on solid supports is discussed in the context of other membrane models. Two native biomembrane systems are presented as examples human erythrocyte ghosts as representatives of eukaryotic cell membranes, and protoplasts from Micrococcus luteus as membranes... 

Keywords Solid-state NMR structure analysis 19F-labeling Membrane-active peptides Native biomembranes Oriented membrane models Antimicrobial peptides... 

Fig. 1 Solid-state NMR structure analysis relies on the 19F-labelled peptides being uniformly embedded in a macroscopically oriented membrane sample, (a) The angle (0) of the 19F-labelled group (e.g. a CF3-moiety) on the peptide backbone (shown here as a cylinder) relative to the static magnetic field is directly reflected in the NMR parameter measured (e.g. DD, see Fig. 2c). (b) The value of the experimental NMR parameter varies along the peptide sequence with a periodicity that is characteristic for distinct peptide conformations, (c) From such wave plot the alignment of the peptide with respect to the lipid bilayer normal (n) can then be evaluated in terms of its tilt angle (x) and azimuthal rotation (p). Whole-body wobbling can be described by an order parameter, S rtlo. (d) The combined data from several individual 19F-labelled peptide analogues thus yields a 3D structural model of the peptide and how it is oriented in the lipid bilayer...

CSA [46]. The local alignment of a peptide segment can thus be described by a C-CF3 vector that protrudes in a well-defined way from the a carbon on the backbone (see Sect. 2.2 on 19F-labelled amino acids). 

Several local labels need to be measured, usually one-by-one in individual samples in the case of 19F-NMR. The combined set of anisotropic NMR parameters then allows one to re-construct the geometry of the entire peptide and to determine its alignment in the membrane, as illustrated in Fig. 1 [35-37, 47, 48]. The only prerequisite is that the 19F-labelled moiety has to be rigidly attached to the peptide backbone, and that the peptide assumes a well-defined secondary structure. Provided that a sufficient number of local orientational constraints can be measured... 

Compared to conventional NMR isotopes (13C, 15N, 2H), 19F-labels cannot be readily placed into proteins in a versatile manner by any biosynthetic expression strategy. Certain auxotrophic bacterial strains can be used to incorporate iso-steric 19F-labelled amino acids (e.g. Fluoro-Phe, Fluoro-Trp, Fluoro-Leu, Fluoro-Ile see Fig. 3), but yields tend to be low. Many other fluoro-organics are toxic if they get converted into fluoroacetic acid, which blocks the enzyme aconitase in the citric... 

Fig. 3 Important 19F-labelled amino acids, (a) Compounds that are wo-steric to native amino acids can be incorporated into proteins biosynthetically, but they possess too many degrees of torsional freedom to be useful for ssNMR structure analysis, (b) In these artificial amino acids the 19F-reporter group is rigidly attached to the peptide backbone. They can be incorporated by solid-phase peptide synthesis, but some problems can arise due to racemisation (4F-Phg, 4CF3-Phg), steric hindrance of coupling (F3-Aib) or HF elimination (fluoro-Ala, F3-Ala). 4F-Phg is additionally problematic due to an ambiguity of the side-chain rotamer. The preferred 19F-labels for ssNMR structure analysis are CF3-Bpg and CF3-Phg (as suitable substitutes for Leu, lie, Met, Val and Ala), as well as F3-Aib and CF3-MePro...

Fig. 8 Representative solid-state 19F-NMR spectra of 19F-labelled gramicidin S (substituted at both Leu positions with 4F-Phg), embedded in macroscopically oriented ghost membranes (left column) and DMPC bilayers (right column) at a peptide-to-lipid ratio of about 1 40 [in (F) the lipid was 1 1 DMPC/cholesterol]. Depending on temperature, the peptide can assume different alignment states, which are strikingly similar in the native membranes and the model bilayers...

Wadhwani P, Strandherg E (2009) Structure analysis of membrane-active peptides using 19F-labeled amino acids and solid-state NMR. In Ojima I (ed) Fluorine in medicinal chemistry and chemical biology. Wiley, Chichester, pp 463-493... 

Although initially mainly of theoretical interest, through-space couplings are now considered an essential element of structure, in particular stereochemical elucidation, and there was an early study in which the fluorine-fluorine TS couplings in 19F-labeled amino acids were used to elucidate the folding of a protein.21... 

NMR can help to monitor energization, see, e.g. [121, 273], especially the levels of 31P-containing metabolites, e.g. [45, 366], enzyme kinetics, compartmentalized intracellular ion activities, the fate of 3H-, 2H-, 13C-, 15N-, or 19F-labeled tracers, e.g. [108,109], 02 tension, compartmentalized redox potential, membrane potential, cell number or cell volume, see [133], and even pH. Major drawbacks are the cost of the equipment, the low intrinsic sensitivity and the interpretation of spectra [430]. 

Structure Analysis of Membrane-Active Peptides Using 19F-labeled Amino Acids and Solid-State NMR... 

This section briefly describes the 19F NMR experiments performed on 19F-labeled peptides, the fundamental NMR parameters that are used to obtain orientational constraints, and the means by which peptide structure is determined from these constraints. 

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