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. 1995 Jul 14;269(5221):192-7.
doi: 10.1126/science.7618079.

Protein folding intermediates: native-state hydrogen exchange

Y Bai  1 , T R Sosnick, L Mayne, S W Englander
Affiliations

Protein folding intermediates: native-state hydrogen exchange

Y Bai et al. Science. .

Abstract

The hydrogen exchange behavior of native cytochrome c in low concentrations of denaturant reveals a sequence of metastable, partially unfolded forms that occupy free energy levels reaching up to the fully unfolded state. The step from one form to another is accomplished by the unfolding of one or more cooperative units of structure. The cooperative units are entire omega loops or mutually stabilizing pairs of whole helices and loops. The partially unfolded forms detected by hydrogen exchange appear to represent the major intermediates in the reversible, dynamic unfolding reactions that occur even at native conditions and thus may define the major pathway for cytochrome c folding.

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Figures

Fig. 1
Fig. 1
Hydrogen exchange behavior of some peptide group NH hydrogens in cytochrome c (oxidized form from horse heart). (A) Slowly exchanging NH’s with low m value (Eq. 3) merge into a common hydrogen exchange isotherm that reveals the GdmCl-enhanced global unfolding reaction [at 50°C; data from (8)]. (B and C) The analogous grouping of hydrogens into lower lying isotherms as their exchange becomes dominated by larger unfolding reactions, promoted by denaturant (30°C) and by temperature. Data are at pD7. These and subsequent curves were fit with equations derived from Eqs. 3 to 5 (11, 41).
Fig. 2
Fig. 2
Hydrogen exchange behavior defining the cooperative reversible unfolding of the amino-terminal (A) and carboxyl-terminal (B) helices. The structural positions of the hydrogens measured are illustrated (C). In these and subsequent hydrogen exchange experiments, oxidized equine cytochrome c in D2O at pD7 and 30°C was used and then analyzed at pD5 in the reduced form by 1D and 2D NMR with the proton assignments from Wand et al. (42) as described (11). Molecular diagrams were obtained with Molscript (43).
Fig. 3
Fig. 3
The cyclic heme-associated structure in native cytochrome c. The hydrogens indicated exhibit protection against exchange in the transient globally unfolded form.
Fig. 4
Fig. 4
Hydrogens of the 60’s helix (A) and the 20 to 35 loop (B) and their common hydrogen exchange isotherm. Dashed lines show the positions of neighboring isotherms. The two cooperative segments and their neighboring structure are illustrated.
Fig. 5
Fig. 5
The large bottom loop isotherm (A) and the small loop isotherm (B). The omega loops in the native protein are illustrated.
Fig. 6
Fig. 6
The cooperative units in cytochrome c, color coded in order of increasing free energy level (red to blue), as follows: the 70 to 85 loop (red), the 36 to 61 loop (yellow), the 20 to 35 loop [green(a)] and 60’s helix [green(b)], the amino-terminal helix [blue(a)] and carboxyl-terminal helix [blue(b)]. Core side chains of the green(a) and green(b) segments make contact beyond the far edge of the heme (17, 18,44).
Fig. 7
Fig. 7
A test for the identities of the different PUFs in terms of the cooperative units that may be folded and unfolded in each. The m value measured for each PUF is plotted against the additional solvent accessible surface (Δarea), relative to the native protein, computed for each possible PUF identity. Each possible partially unfolded form (Px) is identified in terms of the cooperative units that remain folded, noted by the energy-related color code of Fig. 6 (R for red, Y for yellow, G for green, B for blue). Exposed surface calculated for each candidate PUF comes from the newly exposed protein and heme surface plus the cooperative segments that are unfolded, approximated as fully extended. The scale on the right shows the cooperative unit seen to be unfolded in each hydrogen exchange isotherm and the Gibbs free energy for the unfolded state, obtained from the hydrogen exchange isotherm at zero GdmCI concentration (pD7, 30°C). The correlation line drawn is determined by the fact that the unfolding of the amino helix plus carboxyl helix unit (blue) is known to produce the globally unfolded state (U) (11). The ΔG and m parameters relate to the whole molecule PUF and not merely to the individual cooperative unit newly unfolded.
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