KNOWLEDGE-BASED HOMOLOGY MODELING AND EXPERIMENTAL-DETERMINATION OF AMINO-ACID SIDE-CHAIN ACCESSIBILITY BY THE LASER PHOTO CIDNP (CHEMICALLY-INDUCED DYNAMIC NUCLEAR-POLARIZATION) APPROACH IN SOLUTION - LESSONSFROM THE SMALL SIALIDASE OF CLOSTRIDIUM-PERFRINGENS

The success of knowledge-based homology modelling is critically depend ent on the predictive potency of the program structure-based calculati ons, which attempt to translate homologous sequences into three-dimens ional structures, and on the actual relevance of the crystal structure for the protein topology. As quality control, experimental data for s elected parameters of the protein's conformation are required. Using t he crystal structure of the sialidase of Salmonella typhimurium as fra mework for model building of the homologous enzyme from Clostridium pe rfringens, a set of energy-minimised conformers is derived. These prot eins present e.g. Tyr, Trp and His residues with an assessable area on the surface, since the side chains of these amino acid residues are r esponsive to chemically induced dynamic nuclear polarization (CIDNP), monitored by NMR. Hence, as first lesson, a comparative analysis for m odel-derived and experimentally determined values can be performed. Th e second lesson of this study concerns the notable impact of single am ino acid substitutions (Tyr/Phe, Cys/Ser) on the surface accessibility of the CIDNP-reactive amino acid side chains in mutant forms of the s ialidase. Corroborating the predictions from the theoretical calculati ons, the spectra of the engineered mutants reveal marked and non-unifo rm alterations. Thus, the effect of apparently rather conservative ami no acid substitutions on a distinct conformational aspect of this prot ein, even at distant sites, should not be underestimated.