Group epitope mapping considering relaxation of the ligand (GEM-CRL): Including longitudinal relaxation rates in the analysis of saturation transfer difference (STD) experiments

Abstract: In the application of saturation transfer difference (STD) experiments to the study of protein–ligand interactions, the relaxation of the ligand is one of the major influences on the experimentally observed STD factors, making interpretation of these difficult when attempting to define a group epitope map (GEM). In this paper, we describe a simplification of the relaxation matrix that may be applied under specified experimental conditions, which results in a simplified equation reflecting the directly transferred magnetisation rate from the protein onto the ligand, defined as the summation over the whole protein of the protein–ligand cross-relaxation multiplied by with the fractional saturation of the protein protons. In this, the relaxation of the ligand is accounted for implicitly by inclusion of the experimentally determined longitudinal relaxation rates. The conditions under which this “group epitope mapping considering relaxation of the ligand” (GEM-CRL) can be applied were tested on a theoretical model system, which demonstrated only minor deviations from that predicted by the full relaxation matrix calculations (CORCEMA-ST) . Furthermore, CORCEMA-ST calculations of two protein–saccharide complexes (Jacalin and TreR) with known crystal structures were performed and compared with experimental GEM-CRL data. It could be shown that the GEM-CRL methodology is superior to the classical group epitope mapping approach currently used for defining ligand–protein proximities. GEM-CRL is also useful for the interpretation of CORCEMA-ST results, because the transferred magnetisation rate provides an additional parameter for the comparison between measured and calculated values. The independence of this parameter from the above mentioned factors can thereby enhance the value of CORCEMA-ST calculations. [Copyright &y& Elsevier]