The foldon substructure of staphylococcal nuclease

摘要

To search for submolecular foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease under native conditions was studied by a kinetic native-state hydrogen exchange (HX) method. As for other proteins, it appears that staphylococcal nuclease is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measure the equilibrium stability (Delta G(HX)) and the kinetic unfolding and refolding rates (k(op) and k(cl)) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining FIX grouping contains residues, not well separated by their FIX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with a rate constant of 6 x 10(-6) s(-1) and free energy equal to the protein's global stability (10.0 kcal/ mol). It represents part of the 0-barrel, including mutually H-bonding residues in the beta 4 and beta 5 strands, a part of the beta 3 strand that H-bonds to beta 5, and residues at the N-terminus of the alpha 2 helix that is capped by beta 5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native alpha 2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded beta 1-beta 2-beta 3 meander, completing the native beta-barrel, plus an adjacent part of the alpha 1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some normative misfolding interactions. Because the unfolding parameters of many other residues are not determined, it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed. (C) 2007 Elsevier Ltd. All rights reserved.