CONFORMATIONAL-ANALYSIS OF GPA AND GPAP IN AQUEOUS-SOLUTION BY MOLECULAR-DYNAMICS AND STATISTICAL-METHODS

Barnase, an extracellular endoribonuclease from Bacillus amyloliquefac iens, hydrolyses single-stranded RNA. Its very low catalytic activity toward GpN dinucleotides, where N stands for any nucleoside, is marked ly increased when a phosphate is added to the 3'-end, as in GpNp. Here we investigate the conformational properties of GpA and GpAp in solut ion, in order to determine whether differences in these properties may be related to the changes in enzymatic activity. Two independent 1.3 ns molecular dynamics trajectories are generated for each dinucleotide in the presence of explicit water molecules and counter ions. These t rajectories are analysed by monitoring molecular properties, such as t he solvent accessible surface area, the distance and orientation betwe en the bases, the behaviour of torsion angles and formation of intramo lecular H-bonds. To identify relevant correlations between these param eters, statistical techniques, comprising multiple regression, cluster ing and discriminant analysis are used. Results show that GpA has a si gnificant propensity to form folded conformations (similar to 50%), fo stered by a small number of intramolecular H-bonds, whereas GpAp remai ns essentially extended. The latter behaviour seems to he due to an H- bond between the terminal phosphate and adenosine ribose group, which restricts rotation about the adenine Ay angle. We also find that GpA f olding is induced by a concerted motion of specific torsion angles, wh ich is closely coupled to the formation of a network of flexible hydro gen bonds. Finally, on the basis of an expression for barnase K-M, whi ch incorporates the folded/extended conformational equilibria of the d inucleotide substrates, it is argued that our findings on the differen ces between these equilibria, can qualitatively rationalize the experi mentally measured differences in enzymatic properties. (C) 1998 Academ ic Press.