Secondary structure in solution of barwin from barley seed using proton nuclear magnetic resonance spectroscopy

Barwin, a basic protein from barley seed of 125 amino acid residues, has been studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. This protein is closely related to the C-terminal domain of proteins whose synthesis is induced by wounding, the so-called win proteins. These proteins may, therefore, have a role in the defense against fungal attack. Full assignment of the 1H nuclear magnetic resonances has been obtained for 104 amino acid residues, and 18 amino acid spin systems were partially assigned. Sequence-specific assignment using nuclear Overhauser spectroscopy has been achieved for 122 of the 125 residues. This has revealed that the secondary structure of the protein is dominated by a large four-stranded antiparallel beta-sheet consisting of the strands Gln2-Thr9, Lys65-Asn71, Gln77-Arg81, and His113-Val121, a small parallel beta-sheet of the strands Trp48-Cys52 and Asp84-Ala87, which together account for a third of the protein. Sequential effects indicate the presence of three small alpha-helices, Tyr30-Lys38, Leu40-Tyr46, and Thr97-Asp103. The secondary structure in other regions of the sequence is characterized mainly by loops and turns and regions where no regular secondary structure arrangement could be identified. A large number of long-range nuclear Overhauser effects has been identified, and these have been used, together with sequential and intranuclear Overhauser effects, for a calculation of the protein's three-dimensional structure.