Abstract

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 (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 a rate constant of 6 × 10^− 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 H-bonding 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 the α2 helix that 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 the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some nonnative 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.

Keywords: staphylococcal nuclease; protein folding; foldons; hydrogen exchange; kinetic native-state hydrogen exchange

Abbreviations: SNase, staphylococcal nuclease; N, I, and U, native, intermediate, and unfolded states; foldon, cooperative folding/unfolding unit; PUF, partially unfolded form (with some foldons formed and others not formed); HX, hydrogen exchange; NHX, native-state hydrogen exchange; ΔG_HX, unfolding free energy measured by hydrogen exchange; m value, d(ΔG_HX)/d[GdmCl]; EX1, monomolecular exchange where hydrogen exchange rate is equal to the determining structural opening rate; EX2, bimolecular exchange where hydrogen exchange rate is proportional to catalyst concentration and the equilibrium constant of the determining structural unfolding; GdmCl, guanidinium chloride; HSQC, heteronuclear single-quantum coherence; 3D, three-dimensional

Article Outline

Introduction

Results

Equilibrium stability from denaturant melting

Kinetic NHX in pulse-labeling mode

Kinetic NHX in real-time mode

Data analysis

SNase foldon units

Misfolding

Discussion

Protein foldons

SNase foldons

Problems in foldon detection

Misfolding in partially unfolded states

Implications

Materials and Methods

Protein preparation

Global unfolding

NMR assignment

Hydrogen exchange

HX theory

Acknowledgements

Appendix A. Supplementary Data

References