INVOLVEMENT OF LASER PHOTO-CIDNP(CHEMICALLY INDUCED DYNAMIC NUCLEAR POLARIZATION)-REACTIVE AMINO-ACID SIDE-CHAINS IN LIGAND-BINDING BY GALACTOSIDE-SPECIFIC LECTINS IN SOLUTION

For proteins in solution the validity of certain crystallographic para meters can be ascertained by a combination of molecular-dynamics (MD) simulations and NMR spectroscopy. Using the laser photo-CIDNP (chemica lly induced dynamic nuclear polarization) technique as a measure for s urface accessibility of histidine, tyrosine and tryptophan, the spectr a of bovine galectin-1 and Erythrina corallodendron lectin (EcorL) are readily reconcilable with the crystallographic data for these two pro teins. The results emphasise the role of Trp68/Trp69 for carbohydrate binding in bovine galectin-1/chicken galectins and of Trp194 in murine galectin-3. This feature derived from the crystal structure of bovine galectin-1 is maintained in solution for the prototype human homologu e, two avian galectins and the chimera-type murine galectin-3, as the spectra corroborate the CIDNP-inferable spatial parameters of the four calculated models for binding-site architecture. In EcorL, Tyr106/Tyr 108 are constituents of the extended combining pocket, which can be sh ielded in solution by ligand presence. Discrepancies between results f rom modelling and CIDNP measurements concern primarily the lack of rea ctivity of histidine residues for human and avian prototype galectins and of Tyr82/Tyr229 of the plant lectin. Site-directed mutagenesis of EcorL is assumed to provide information on the role of a certain resid ue for functional aspects, When single-site mutants of EcorL ([Ala106] EcorL [Ala108]EcorL, [Ala229]EcorL) were subjected to molecular-dynami cs (MD) simulations, the apparent surface accessibilities even of spat ially separated amino acid side chains could non-uniformly be affected . This conclusion is supported by the assessment of the spectra for th e mutant proteins. On the basis of these CIDNP-results modelling of th e binding-site architecture of the lectin indicates the occurrence of notable alterations in the orientation of Tyr106/Tyr108 phenyl rings. The implied potential effect of single-site mutations on conformationa l features of a protein will deserve attention for the interpretation of studies comparing wild-type and mutant proteins.