THE NMR SOLUTION STRUCTURE OF THE MUTANT ALPHA-AMYLASE INHIBITOR [R19L]TENDAMISTAT AND COMPARISON WITH WILD-TYPE TENDAMISTAT

The recombinant mutant a-amylase inhibitor [R19L]Tendamistat, with Arg 19 replaced by Leu, was prepared and its NMR solution structure determ ined. Based on complete sequence-specific H-1-NMR assignments, 845 nuc lear Overhauser effect upper-distance constraints and 156 dihedral ang le constraints were collected using two-dimensional homonuclear H-1-NM R experiments. The structure was calculated with the program DIANA, us ing the redundant dihedral angle constraints strategy for improved con vergence. For restrained energy minimization, the programs FANTOM and AMBER were used. The wild-type NMR solution structure was similarly re calculated from the previously published input of conformational const raints [Kline, A., Braun, W. and Wuthrich, K. (1988) J. Mol. Biol. 204 , 675-724]. For each protein a group of 20 conformers represents a wel l-defined solution structure, with average root-mean-square-distance v alues for the backbone atoms of the individual conformers relative to the mean coordinates of 50 pm. The two structures are nearly identical to each other and to the previously published Tendamistat structures in solution and in crystals. The only significant difference is strict ly localized near the single amino acid substitution in the presumed a ctive site -Trp18-Arg(Leu)-Tyr- i.e. Leu19 and Tyr20 are more precisel y defined in the solution structure of [R19L]Tendamistat than the corr esponding residues Arg19 and Tyr20 in wild-type Tendamistat.