CARBOHYDRATE-PROTEIN INTERACTION STUDIES BY LASER PHOTO CIDNP NMR METHODS

The side chains of tyrosine, tryptophan and histidine are able to prod uce CIDNP (Chemically Induced Dynamic Nuclear Polarization) signals af ter laser irradiation in the presence of a suitable radical pair-gener ating dye. Elicitation of such a response in proteins implies surface accessibility of the respective groups to the light-absorbing dye, In principle, this technique allows the monitoring of the effect of ligan d binding to a receptor and of site-directed mutagenesis on conformati onal aspects of any protein if CIDNP-reactive amino acids are involved . The application of this method in glycosciences can provide insights into the protein-carbohydrate interaction process, as illustrated in this initial model study for several N-acetyl-glucosamine-binding lect ins of increasing structural complexity as well as for a wild type bac terial sialidase and its mutants. Experimentally, the shape and intens ity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When the carbohydrate is bound, CIDNP signa ls of side chain protons of tyrosine, tryptophan or histidine residues can be broadened and of reduced intensity. This is the case for hevei n, pseudo-hevein, the four hevein domains-containing lectin wheat germ agglutinin (WGA) and the cloned B-domain of WGA 1 (domB) representing one hevein domain. This response indicates either a spatial protectio n by the ligand or a ligand-induced positioning of formerly surface-ex posed side chains into the protein's interior part, thereby precluding interaction with the photo-activated dye. Some signals of protons fro m the reactive side chains can even disappear when the lectin-ligand c omplexes are monitored. The ligand binding, however, can apparently al so induce a conformational change in a related lectin that causes the appearance of a new signal, as seen for Urtica dioica agglutinin (UDA) which consists of two hevein domains. Additionally, the three CIDNP-r eactive amino acids are used as sensors for the detection of conformat ional changes caused by pH variations or by deliberate amino acid exch anges, as determined for the isolectins hevein and pseudo-hevein as we ll as for the cloned small sialidase of Clostridium perfringens and tw o of its mutants. Therefore, CIDNP has proven to be an excellent tool for protein-carbohydrate binding studies and can be established in gly cosciences as a third biophysical method beside X-ray-crystallography and high-resolution multidimensional NMR studies which provides reliab le information of certain structural aspects of carbohydrate-binding p roteins in solution.