The nuclear-magnetic-resonance solution structure of the mutant alpha-amylase inhibitor [R19L] Tendamistat and comparison with wild-type Tendamistat.

Abstract

The recombinant mutant alpha-amylase inhibitor [R19L]Tendamistat, with Arg19 replaced by Leu, was prepared and its NMR solution structure determined. Based on complete sequence-specific 1H-NMR assignments, 845 nuclear Overhauser effect upper-distance constraints and 156 dihedral angle constraints were collected using two-dimensional homonuclear 1H-NMR experiments. The structure was calculated with the program DIANA, using the redundant dihedral angle constraints strategy for improved convergence. For restrained energy minimization, the programs FANTOM and AMBER were used. The wild-type NMR solution structure was similarly recalculated from the previously published input of conformational constraints [Kline, A., Braun, W. & Wüthrich, K. (1988) J. Mol. Biol. 204, 675-724]. For each protein a group of 20 conformers represents a well-defined solution structure, with average root-mean-square-distance values 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 strictly localized near the single amino acid substitution in the presumed active site -Trp18-Arg(Leu)-Tyr-, i.e. Leu19 and Tyr20 are more precisely defined in the solution structure of [R19L]Tendamistat than the corresponding residues Arg19 and Tyr20 in wild-type Tendamistat.