The nature of protein folding pathways

Abstract

How do proteins fold, and why do they fold in that way? This Perspective integrates earlier and more recent advances over the 50-y history of the protein folding problem, emphasizing unambiguously clear structural information. Experimental results show that, contrary to prior belief, proteins are multistate rather than two-state objects. They are composed of separately cooperative foldon building blocks that can be seen to repeatedly unfold and refold as units even under native conditions. Similarly, foldons are lost as units when proteins are destabilized to produce partially unfolded equilibrium molten globules. In kinetic folding, the inherently cooperative nature of foldons predisposes the thermally driven amino acid-level search to form an initial foldon and subsequent foldons in later assisted searches. The small size of foldon units, ∼ 20 residues, resolves the Levinthal time-scale search problem. These microscopic-level search processes can be identified with the disordered multitrack search envisioned in the "new view" model for protein folding. Emergent macroscopic foldon-foldon interactions then collectively provide the structural guidance and free energy bias for the ordered addition of foldons in a stepwise pathway that sequentially builds the native protein. These conclusions reconcile the seemingly opposed new view and defined pathway models; the two models account for different stages of the protein folding process. Additionally, these observations answer the "how" and the "why" questions. The protein folding pathway depends on the same foldon units and foldon-foldon interactions that construct the native structure.

Keywords: hydrogen exchange; protein folding; protein structure.

Fig. 1.

(A) The classical view of a defined folding pathway, and (B) the new view of multiple routes through a funneled landscape. Reprinted with permission from ref. . Dashed line in A illustrates the insertion of an optional error-dependent kinetic barrier, which can affect some population fraction and not others and thus mimic multipathway folding.

Fig. 2.

Initial HX NMR pulse labeling results for Cyt c (24). A brief D to H labeling pulse imposed after various folding times was used to track the increasing protection (decreasing H-labeling) of individual residues and the segments that they represent. The results suggested early formation of a native-like N/C bihelical folding intermediate. Baldwin’s review (21) noted the kinetic asynchrony, with the N- and C-terminal helical segments in different molecules folding at different rates. Later work shows that the asynchrony is caused by protein aggregation and by HX pulse breakthrough due to back-unfolding of the transiently populated intermediate during the H-labeling pulse (50 ms).

Fig. 3.

Pulse labeling HX MS results for maltose binding protein (29). (A) The time-dependent folding (HX protection) of 116 highest-quality MBP peptide fragments representing different protein regions. Black kinetic curves show the slow time course for folding of peptide fragments that are most protected in the initial collapse. (B–D) Representative HX-labeled MS fragments from different protein regions (colored) define the separate folding steps, display their concerted two-state nature, measure their formation rates, and show that the entire protein population (>95%) experiences the same steps. (E) The course of folding. On dilution from denaturant into folding conditions, MBP rapidly collapses into a heterogeneous polyglobular state (SAXS envelope reconstruction in gray) with widespread low level HX protection, then slowly folds (kinetic curves in A) through an initial native-like intermediate (blue, τ = 7 s) and later kinetically unresolved steps (green, gray, red; τ ∼60 s to 120 s; fastest green segments shown in C and E). Mutations known to greatly slow folding (stars) are all within the 7 s intermediate.

Fig. 4.

Pulse labeling HX MS results for Ribonuclease H (30). (A and B) Kinetic curves for time-dependent HX protection of peptide fragments that define the blue, green, yellow, and red foldons. (C–F) HX MS pulse labeling results for representative peptide fragments show the time course and two-state concerted nature of foldon folding steps, and that the entire protein population (>95%) experiences the same sequence of concerted steps in a single dominant pathway. The yellow foldon does not reach complete protection because of partial labeling due to back-unfolding during the 10-ms labeling pulse, which helps to distinguish the yellow foldon from the green foldon, along with the small difference in their formation rates seen in the renormalized kinetic phases (A, Inset).

Fig. 5.

Initial equilibrium native state HX NMR results for Cyt c (47). (A–D) HX rates of many individual Cyt c residues, measured by NMR as a function of low levels of added denaturant far below the melting transition, are plotted in terms of the free energy of the exposure reaction that determines each amide HX rate. HX governed by a small local fluctuation is insensitive to denaturant and produces a horizontal curve. HX determined by a large unfolding reaction is sharply promoted by denaturant and can come to dominate the exchange of the residues that it exposes. The residues that join each cooperative unfolding (large slope) specify the identity of that unfolding unit. The intercept of each HX isotherm defines the free energy level of each PUF at zero denaturant; the slope relates to its surface exposure. These data identified four large unfolding units (foldons), coded as blue, green, yellow, and red. The less definitive red foldon and the infrared foldon not seen here (gray in Fig. 6) were better defined in later work. (E) The free energy levels of the PUFs produced by the individual cooperative unfolding reactions place them on a free energy ladder. The data in A–D specify the identity of each foldon unfolding unit but do not specify the complete PUF produced by each unfolding. Therefore, one cannot tell whether the foldons unfold independently or sequentially or in some other manner. A series of stability labeling experiments defined the PUFs shown (far right). They constitute a stepwise unfolding and refolding pathway, as in Fig. 1A.

Fig. 6.

The foldon construction of Ribonuclease H and Cyt c. The order of folding is blue, green, yellow, and red, and finally gray for the large bottom Cyt c loop.