A protein folding pathway with multiple folding intermediates at atomic resolution

Abstract

Using native-state hydrogen-exchange-directed protein engineering and multidimensional NMR, we determined the high-resolution structure (rms deviation, 1.1 angstroms) for an intermediate of the four-helix bundle protein: Rd-apocytochrome b562. The intermediate has the N-terminal helix and a part of the C-terminal helix unfolded. In earlier studies, we also solved the structures of two other folding intermediates for the same protein: one with the N-terminal helix alone unfolded and the other with a reorganized hydrophobic core. Together, these structures provide a description of a protein folding pathway with multiple intermediates at atomic resolution. The two general features for the intermediates are (i) native-like backbone topology and (ii) nonnative side-chain interactions. These results have implications for important issues in protein folding studies, including large-scale conformation search, -value analysis, and computer simulations.

Fig. 1.

The use of NHX-directed protein engineering to populate PUF1 of Rd-apocyt b₅₆₂. (a) Representative NHX results for Rd-apocyt b₅₆₂ (18). Two color-coded amide protons corresponding to the region of the structural unit in the structure of the native state are shown for each structural unit. The horizontal dashed lines indicate the unfolding free energy of PUF1, PUF2, and the unfolded state at zero denaturant. (b) Structures of the PUFs derived from the complete NHX results. (c) Gly mutations that substitute hydrophobic core residues in the unfolded regions (red and green) of PUF1 destabilize the native state and PUF2 without affecting the stability of the unfolded state and PUF1. Therefore, PUF1 becomes the most stable species. The dashed line indicates that PUF2 and N are high-energy species after the Gly mutations. (d) Structural image of the mutated residues in the native structure of Rd-apocyt b₅₆₂ for populating PUF1 (59). The side chains of these residues are shown in Corey–Pauling–Kolton models. The Trp at position 7 was mutated to Asp. All other residues were changed to Gly.

Fig. 2.

Dynamic properties of the PUF1 mimic. (a) The ¹H—¹⁵N, the heteronuclear single-quantum correlation spectrum of the PUF1 mimic. (b) Amide ¹⁵N dynamics of the PUF1 mimic at 15°C and pH 4.8. □, T₂ values; •, NOEs.

Fig. 3.

NMR structure of the PUF1 mimic. (a) Backbone conformation, in which 10 NMR structures are superimposed on the Cα carbons from residues 24 to 94. (b) Some hydrophobic side chains.

Fig. 4.

Comparison of the ribbon structures of PUF1 (a), PUF2 (b), N* (c), and N (d). Representative hydrophobic residues are shown in Corey–Pauling–Kolton models. The four helices are labeled with I, II, III, and IV from the N terminus to the C terminus in a.

Fig. 5.

A putative folding pathway with multiple intermediates at atomic resolution for Rd-apocyt b₅₆₂. The specific discrete intermediates form sequentially. The order for folding is defined by the nativeness of the intermediates. PUF1, PUF2, N*, and N are represented by 10 structures determined by NMR methods. Aromatic side chains for residues Y101 and Y105 (purple) in N* and N are shown to illustrate their different conformations in the two structures. Note that N* is observed at 2.8 M urea. Its existence in water needs to be demonstrated further.