3-DIMENSIONAL STRUCTURE IN SOLUTION OF BARWIN, A PROTEIN FROM BARLEY SEED

The solution structure of a 125-residue basic protein, barwin, bas been determined using H-1 nuclear magnetic resonance spectroscopy. This protein is closely related to domains in proteins encoded by wound-induced genes in plants. Analysis of the H-1 nuclear Overhauser spectrum revealed the assignment of more than 1400 nuclear Overhauser effects. Twenty structures were calculated based on 676 nontrivial distance restraints, 152 torsion angle restraints (92-phi, 56-chi-1, and 4-omega for proline), and stereospecific assignments of 38 chiral centers, using distance geometry, simulated annealing, and restrained energy minimization. None of the distance restraints was violated by more than 0.5 angstrom in any of the 20 structures, and none of the torsion angle restraints was violated by more than 1-degrees in any of the structures. The RMS difference between the calculated and target interproton distance restraints is 0.033 angstrom, and the average atomic RMS differences between the 20 structures and their geometric average are 1.23 angstrom for backbone atoms and 1.73 angstrom for all heavy atoms. The dominating structural feature of the protein is a well-defined four-stranded antiparallel beta-sheet, two parallel beta-sheets packed antiparallel to each other and four short alpha-helices. The binding site of barwin to the tetramer N-acetylglucosamine has been qualitatively investigated, and the dissociation constant of the complex has been determined using one-dimensional H-1 nuclear magnetic resonance spectroscopy.