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. 2013 May 7;110(19):7684-9.
doi: 10.1073/pnas.1305887110. Epub 2013 Apr 19.

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

Wenbing Hu  1 , Benjamin T Walters, Zhong-Yuan Kan, Leland Mayne, Laura E Rosen, Susan Marqusee, S Walter Englander
Affiliations

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

Wenbing Hu et al. Proc Natl Acad Sci U S A. .

Abstract

The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on native-like interfoldon interactions orders the pathway.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The folding of RNase H monitored by circular dichroism. (A) A burst-phase intermediate (Icore) is formed within the dead time of the experiment (manual mixing) followed by slower folding (30 s) to the native state (0.6 M urea, 10 °C). (B) The equilibrium urea melt (black circles), the amplitude of the burst phase (diamonds), and the observable phase (white circles). (C) Folding and unfolding rates (chevron plot) at 10 °C (black trace) compared with the published fit at 25 °C (gray trace).
Fig. 2.
Fig. 2.
The HX MS pulse-labeling experiment. (A) Denaturant-unfolded fully deuterated protein is diluted into folding conditions (mixer 1). After some folding time, D-to-H exchange at still-exposed sites is induced by a brief high-pH pulse (mixer 2) and then is quenched to low pH where HX is very slow (mixer 3). Online proteolysis cuts the protein into many overlapping peptide fragments, and the peptides are separated by LC and MS. (B) The 228 unique peptides used in this work, identified and characterized in the MS data by the ExMS program (12), and plotted as a function of amino acid residue.
Fig. 3.
Fig. 3.
(A–D) Illustrative MS spectra versus folding time. Peptides shown (also see SI Appendix) cover each helical segment plus some neighboring sequence in the native protein. The top and bottom frames show control experiments in which the unfolded and native proteins were subjected to the same labeling pulse and analysis. Fitted envelopes separate the fractional populations of the unfolded, intermediate, and native state present at the time of the labeling pulse. Deuterons on side chains and the first two residues of each peptide are lost during sample preparation. The subpeaks within each isotopic envelope are caused by the natural abundance of 13C (∼1%) convolved with the carried number of deuterons. A leftward drift in folded peptide mass at long folding times (D) occurs because not-yet-protected sites are exposed to D-to-H exchange during the prepulse folding period (pH 5, 10 °C). (E and F) The time dependence for the formation of the protected state of different protein regions, color coded to match the RNase H foldon units in Fig. 5. (Inset) The unblocked folding phase of the yellow curves is renormalized to 100% to allow direct comparison with the folding time of the green segment. For this comparison, the experiment was replicated in triplicate, and only the highest-precision peptides were used. The green and yellow segments fold in detectably different phases. Peptides are identified in SI Appendix, Fig. S1.
Fig. 4.
Fig. 4.
Protected D label at residue resolution. Site-resolved D occupancy was computed from MS data for many overlapping peptides after different folding times: at the earliest folding time in a competition folding versus labeling experiment (Top); after different prepulse folding times (Middle); and for a native protein control (Bottom). Dots through the native frame indicate amide H-bonds to main chain (open) and to side chains (filled) (PDB ID: 1F21). Individually resolved residues are shown in red. Switchable residues not distinguished because of inadequate peptide coverage are in blue and are connected by a dashed line. Small white spaces indicate absent amides because of proline or high probability protease cut sites. Regions in gray indicate segments that are in transition from unfolded to folded conformations and that produce bimodal MS spectra that cannot be analyzed for site resolution. Positions of helices A–E and β-strands 1–5 in the native protein are shown at the top, color coded to match their foldon identities (Fig. 5). Low D retention in the D-to-H labeling pulse indicates an equilibrium protection factor <10; full D retention indicates a protection factor >100 or kop <20 s−1. A nonzero D label in the absence of protection reflects the 6% D2O in the labeling pulse.
Fig. 5.
Fig. 5.
RNase H folding pathway. (A) RNase H foldon units. (B) The macroscopic folding reaction is well represented by a conventional free energy diagram.
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