The Foldon Substructure of Staphylococcal Nuclease

Abstract

To search for submolecular protein foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease (SNase) under native conditions was studied by a kinetic native state hydrogen exchange (NHX) method. As for other proteins, it appears that SNase 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 measures the equilibrium stability (ΔG 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 HX grouping contains residues, not well separated by their HX 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 rate constant of 6x10 −6 s −1 and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the β-barrel including mutually Hbonding residues in the β4 and β5 strands, a part of the β3 strand that H-bonds to β5, and residues at the N-terminus of theα2 helix which is capped by β5. 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 α2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded β1-β2-β3 meander, completing the native β-barrel, plus an adjacent part of the α1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although structure of the partially unfolded forms (PUFs) closely mimics the native organization, four residues indicate the presence of some non-native 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.

Protein folding and misfolding: mechanism and principles

“…Moreover, entire Ω-loops act as concerted unfolding units (Hoang et al 2002;Krishna et al 2003b), unsurprisingly so, because they are internally packed self-contained structures (Leszczynski & Rose, 1986). β-structures tend to break up into smaller separately cooperative units (Chamberlain et al 1996;Yan et al 2002Yan et al , 2004Bédard et al 2008). …”

Protein folding: Independent unrelated pathways or predetermined pathway with optional errors

“…Fig. 1A Inset includes the unfolding rate measured at zero denaturant by kinetic native-state hydrogen exchange (24). The curvature is due to the changing dominance of a succession of on-pathway barriers, indicating the presence of several on-pathway intermediates.…”

“…Kamagata et al (12) used direct and interrupted folding experiments to show that the fluorescence changes observed in SNase folding directly measure acquisition of the native state. This occurs because the populated kinetic intermediates of SNase have fluorescence that is identical to the unfolded state, and the C-terminal segment that harbors the lone SNase tryptophan, Trp-140, is the last to fold (24).…”

“…1). Kinetic principles dictate that the three kinetic phases require the significant population of four states, namely U, N, and two (Inset) Unfolding rate at zero denaturant measured by kinetic native-state hydrogen exchange (24). (B) Kinetic folding data for native-state formation at zero denaturant, shown at the earliest folding times to exhibit the lag phase.…”

Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis

“…Results for 81 amino acids are within threefold of the measured NMR rate, 16 are within 10-fold, and 2 are outliers. The MS and NMR data were measured in different years under different solution conditions by different operators (41,42) and corrected to the common scale of HX protection (1). These factors appear to dominate the variance observed (e.g., compare the calculational accuracy seen in Fig.…”