Protein folding: the stepwise assembly of foldon units

Abstract

Equilibrium and kinetic hydrogen exchange experiments show that cytochrome c is composed of five foldon units that continually unfold and refold even under native conditions. Folding proceeds by the stepwise assembly of the foldon units rather than one amino acid at a time. The folding pathway is determined by a sequential stabilization process; previously formed foldons guide and stabilize subsequent foldons to progressively build the native protein. Four other proteins have been found to show similar behavior. These results support stepwise protein folding pathways through discrete intermediates.

Fig. 1.

Cyt c color-coded to indicate its component foldon units.

Fig. 2.

Equilibrium NHX results color-coded as in Fig. 1. Data are for WT Cyt c (12), recombinant pWT Cyt c, and two pWT variants [pDr 7.0 and 30°C (WT) or 20°C (pWT)]. The amide NHs shown in gray have residual protection in the unfolded forms.

Fig. 3.

Stability labeling results showing the change in the free energy level of the Cyt c unfolded states caused by selective perturbations. Compared are: reduced and oxidized WT Cyt c (A); the effect of a Glu-62-Gly mutation in pWT Cyt c (B); and the effect of a Glu-66 -Ala mutation in pWT Cyt c (C). The free energy levels compare values for the isotherm marker protons at zero denaturant. The nested-yellow unit is omitted in these comparisons; its protons exchange too rapidly to measure under the conditions used.

Fig. 4.

HX pulse-labeling results. Unfolded deuterated WT Cyt c (pDr 7.5, 4.2 M GdmCl, 10°C) was refolded by denaturant dilution (pH 6, 10°C, 0.23 M GdmCl, H₂O). A kinetic intermediate, blocked by a histidine to heme misligation barrier, was H labeled in a 50-ms, high-pH pulse. The refolded native protein was analyzed by 2D NMR. The black dashed curves indicate the labeling expected for each amide in the absence of HX protection. The colored curves fit the labeling actually obtained within the intermediate alone (EX2 and EX1) after correction for extraneous effects (3, 16).

Fig. 5.

Kinetic NHX results. Predeuterated native WT Cyt c was exposed to a 75-ms H labeling pulse at high pH and 20°C, then quenched to slow HX conditions and analyzed by 2D NMR (20). The labeling of the foldon markers is shown in bold. The black curve depends on the rate for formation of a stable high pH intermediate (measured by CD and fluorescence) and shows the EX1 labeling that would be obtained during the 75-ms pulse caused by unfolding to this state. Comparison of the EX1 labeling of the markers for the different foldons at given pH values shows that the kinetic order for unfolding of the different foldon units is first red, then yellow, then green, and finally blue.

Fig. 6.

Foldon behavior in other proteins. Published NHX results for apo Cyt b₅₆₂ (22) and ribonuclease H (27) and sulfhydril reactivity results for triosephosphate isomerase (TIM) (29) are summarized. [Reproduced with permission from ref. (Copyright 2002, American Chemical Society; ref. (Copyright 1996, Nature Publishing Group, www.nature.com); and ref. (Copyright 2002, Elsevier Science Ltd.).]

Fig. 7.

Graphical contact map for the different foldons in Cyt c and the residue contacts that connect them in the native protein (39).