TOWARD SOLVING THE FOLDING PATHWAY OF BARNASE - THE COMPLETE BACKBONE C-13, N-15, AND H-1-NMR ASSIGNMENTS OF ITS PH-DENATURED STATE

The structures of the major folding intermediate, the transition state for folding, and the folded state of barnase have been previously characterized. We now add a further step toward a complete picture of the folding of barnase by reporting the backbone N-15, C-13, and H-1 NMR assignments for barnase unfolded at pH 1.8 and 30 degrees C. These assignments, which were obtained from a combination of heteronuclear magnetization transfer and backbone triple-resonance NMR experiments, constitute the first stage in the structural characterization of this denatured state by NMR. Interresidue nuclear Overhauser effect contacts and deviations from H-1 random-coil chemical shifts provide evidence for stable residual structure. The structured regions span residues in the native protein that contain its major alpha-helix and central strands of the beta-sheet. Earlier experiments have shown that these regions are predominantly intact in the major folding intermediate and that their docking is partly rate determining in folding.