Branching in the sequential folding pathway of cytochrome c

Abstract

Previous results indicate that the folding pathways of cytochrome c and other proteins progressively build the target native protein in a predetermined stepwise manner by the sequential formation and association of native-like foldon units. The present work used native state hydrogen exchange methods to investigate a structural anomaly in cytochrome c results that suggested the concerted folding of two segments that have little structural relationship in the native protein. The results show that the two segments, an 18-residue omega loop and a 10-residue helix, are able to unfold and refold independently, which allows a branch point in the folding pathway. The pathway that emerges assembles native-like foldon units in a linear sequential manner when prior native-like structure can template a single subsequent foldon, and optional pathway branching is seen when prior structure is able to support the folding of two different foldons.

Figure 1.

(A) Cyt c structure (1HRC.pdb; [Bushnell et al. 1990] and MOLSCRIPT [Kraulis 1991]), color-coded to indicate the foldon units previously identified by HX experiments and ranked in spectral order of decreasing ΔG_HX. The five foldons are blue (N- and C-terminal α-helices docked against each other), green (60s helix and 19–36 Ω-loop), yellow (37–39:58–61, a short two-stranded antiparallel β-sheet), red (71–85 Ω-loop), and infrared (40–57 Ω-loop) (Bai et al. 1995; Milne et al. 1999; Krishna et al. 2003b; Maity et al. 2005). (B) Illustration of NHX experiments showing the denaturant dependence of all of the amide hydrogens in the green helix and the measurable green loop hydrogens (oxidized WT equine Cyt c at pDr 7, 30°C); (Bai et al. 1995; Milne et al. 1999). Native Cyt c was placed into D₂O and the H/D exchange of individual amides was measured by recording 2D NMR spectra in time. The experiment was repeated with increasing concentrations of GdmCl but still far below the melting transition (C_m = 2.75 M GdmCl). The ΔG_HX for the opening reaction that determines the HX of each amide was calculated as in Materials and Methods. With increase in denaturant concentration, a sizable unfolding reaction, represented by Leu68, is enhanced and comes to dominate the exchange of all of the green helix amide hydrogens. The measurable green loop hydrogens appear to merge into the same HX isotherm as the green helix, suggesting that they unfold together (except for His33, which is protected by residual structure in the unfolded state). Color-coded dashed lines indicate the positions of other HX isotherms that identify the other foldon units in panel A.

Scheme 1.

Figure 2.

Stability labeling results that test the sequential unfolding nature of the individual Cyt c foldons. NHX experiments were done at pDr 7, 20°C. The effect of each stabilizing or destabilizing perturbation is shown in terms of the change in ΔG_HX measured from the marker protons for each foldon. ΔΔG_HX of the targeted foldon in each case is shown in boldface. Numerical values are in kilocalories per mole. pWT refers to recombinant pseudo-wild-type Cyt c (H26N, H33N) (Rumbley et al. 2002). (A) The E62G mutant compared to pWT Cyt c (Maity et al. 2004). The direct effect of the E62G mutation, which deletes a stabilizing ion pair, is focused on the yellow foldon. The fast exchanging protons in the infrared loop were measured at pDr 5.4. (B) Comparison of reduced and oxidized Cyt c (Xu et al. 1998; Krishna et al. 2003b, 2006). Reducing the heme iron increases the stability of the Met80 sulphur to heme iron bond by 3.2 kcal/mol. The red loop is destabilized by the same amount (the S–Fe bond breaks when red unfolds). Reduced Cyt c is additionally stabilized due to neutralization of the buried heme iron charge, which shows up only in the global blue unfolding. (C) Stability changes between pDr 7 and 4.5. The green loop stability is indicated by the green dashed lines, taken from the data for Leu32 in ▶. The value at pDr 7 represents a minimum estimate for the ΔG_HX of the green loop unfolding (indicated by an upward arrow) since Leu32 exchanges more rapidly (with lower ΔG_HX) than for an unfolding marker. Low pH tends to protonate two buried groups when they are exposed in transient unfolding reactions (His26 and heme propionate 7). This has two stability labeling effects—on the infrared loop, which is supported by hydrogen bonds with the His26 and heme propionate, and an additional effect on the green loop, which buries His26. At pDr 4.5, the marker protons of three foldons (red, yellow, and green loop) have identical ΔG_HX within 0.2 kcal/mol, but are shown vertically displaced in the figure for clarity.

Figure 3.

(A) HX data for the measurable green loop amides at pDr 7 and 4.5 except for His33 (pDr 5.0 shown), for which the normal cross-peak disappears at lower pH due to side chain titration. The expected low pDr slowing of all of these amides (10-fold per pH unit) is largely compensated by the pDr-dependent decrease in foldon stability (K _op). HX rates in such well-determined cases are accurate to about 10% so that ΔG_HX values, calculated from the logarithm of the measured HX rates, are accurate to better than 0.1 kcal/mol. (B) ΔG_HX for the measurable green loop hydrogens of oxidized WT Cyt c. Decreasing pH acts as a destabilant to promote a large unfolding reaction. At neutral pH, the four measurable sequentially distant amides in the green loop exchange through pH-independent unfolding reactions. At lower pH, their ΔG_HX values tend to merge into a single isotherm, reflecting a common unfolding reaction. (C) ΔG_HX for amides in the green helix of oxidized WT Cyt c as f(pH). The green helix marker (Leu68) registers a smaller ΔG_HX change with pH than the green loop. (D) ΔG_HX for the green loop hydrogens in pWT Cyt c (H26N, H33N) as f(pH). Unlike WT Cyt c (panel B), the green loop hydrogens are unaffected by reduced pH, identifying His26 as the major source of the green loop destabilization in WT Cyt c.

Scheme 2.

Figure 4.

Sequential unfolding and refolding pathway of Cyt c. Pathway steps are determined by the intrinsically cooperative protein foldons. The pathway order is determined by the sequential stabilization mechanism in which previously formed structure templates the formation of and stabilizes subsequent foldons, all in the native context, to progressively build the target native structure.