Group epitope mapping

STD experiments allow for a mapping of the binding epitope (group epitope mapping, GEM) [25]. The degree of saturation of ligand resonances depends on the distance of the protons involved. Protons in close proximity to the target molecule are saturated to a... 

Mayer, M. and Meyer, B., Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J Am Chem Soc, 2001,... 

The apparent epitope map based on STDs can exhibit rather significant dependence on which particular protein proton(s) is being saturated. For example, saturation of resolved methyl proton resonances from two separate residues may result in STD spectra with different relative and/or absolute intensities. Similarly, saturation of a specific tyrosine ring proton vs. a specific methyl group can result in different STD spectra. 

Fig. 5 Effect of varying relaxation delays between on- and off-resonance experiments in STD NMR experiments, a Experimental setnp for interleaved measnrements in STD NMR spectroscopy, n represents the nnmber of scans. The inter-scan delay Adi is varied while keeping on- and off-resonance freqnencies constant at -4 and -t300 ppm, respectively, b The resulting STD effects for the 0-methyl group of a-L-Fuc-O-methyl in the presence of RHDV VLPs. The equation that was used for non-linear least squares data fitting is based on the saturation recovery experiment [98], With Ti(iig) = 0.91 s as measured independently (unpublished results) and a Monte Carlo error estimation yields Ti(virus) = 10.06 0.41 s. This value does not directly correspond to a Tl relaxation time of the virus protons, because other factors also influence the observed relaxation [99]. According to these findings a relaxation delay Adi = 25 s was employed in all STD experiments. This results in a recovery of 92% of the virus resonance, and thereby reduces errors in epitope mapping that are introduced otherwise by non-homogeneous recovery of the binding site.

In another paper from the same group (40), a new solution-based approach for linear epitope mapping based on ACE/MS is demonstrated using beta-endorphin as a model substance. The procedure can briefly be described... 

Fig. 11 The binding epitope of anti-lS5 was mapped through STD-NMR experiments [101]. The percentages drawn in the structure indicate the level of transfer of magnetization to a particular hydrogen relative to transfer received by the Af-acetyl methyl group. Large percentages were identified on the benzamide and the nitroindole, thus rationalizing the enhanced affinity of anti-lSS...

This technology has been adopted by many groups and many successes have been reported in experimental animals the first MAPs consisted of a single type of peptide epitope but the availability of orthogonally protected side groups of lysine allows the assembly of MAPs from which different peptide sequences can be synthesised. 

Figure 3. Schematic representation of a tetravalent MAP formed by the addition of a lysine residue to each of the two primary amine groups of a central lysine (K) residue, an airangement that provides four primary amine groups from which separate epitopes can be synthesised. The right hand schematic is an energy minimised molecular model of the trivalent lysine core indicating the alpha and epsilon amino groups to which individual epitopes are added.

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