3-DIMENSIONAL SOLUTION STRUCTURE AND C-13 ASSIGNMENTS OF BARSTAR USING NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

We present the high-resolution solution structure and C-13 assignments of wild-type barstar, an 89 amino acid residue polypeptide inhibitor of barnase, derived from heteronuclear NMR techniques. These were obta ined from measurements on unlabeled, uniformly N-15- and C-13/N-15-lab eled, and 10% C-13-labeled barstar samples that have both cysteines (a t positions 40 and 82) fully reduced. In total, 30 structures were cal culated by hybrid distance geometry-dynamical simulated annealing calc ulations. The atomic rms distribution about the mean coordinate positi ons is 0.42 Angstrom for all backbone atoms and 0.90 Angstrom for all atoms. The structure is composed of three parallel alpha-helices packe d against a three-stranded parallel beta-sheet. A more poorly defined helix links the second beta-strand and the third major alpha-helix. Th e loop involved in binding barnase is extremely well defined and held rigidly by interactions from the main body of the protein to both ends and the middle of the loop. This structure will be used to aid protei n engineering studies currently taking place on the free and bound sta tes of barstar and barnase.