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1.
Figure 4.

Figure 4. From: Branching in the sequential folding pathway of cytochrome c .

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.

Mallela M.G. Krishna, et al. Protein Sci. 2007 September;16(9):1946-1956.
2.
Scheme 2.

Scheme 2. From: Branching in the sequential folding pathway of cytochrome c .

These results point to the modified reaction Scheme 2, where G and g refer to the native and unfolded green helix, and G′ and g′ refer to the native and unfolded green loop. The new feature compared to Scheme 1 is that the pathway can optionally branch at the steps where the two green units are able to fold, or unfold, separately. Otherwise the folding pathway maintains the same stepwise sequential nature, adding or subtracting one native-like foldon unit at a time to transit from one PUF to the next.

Mallela M.G. Krishna, et al. Protein Sci. 2007 September;16(9):1946-1956.
3.
Scheme 1.

Scheme 1. From: Branching in the sequential folding pathway of cytochrome c .

Results from a number of stability labeling experiments show that the foldons unfold in a sequential pathway manner to produce a series of partially unfolded forms (PUFs) that occupy increasingly higher free energy levels in the energy landscape (Xu et al. 1998; Maity et al. 2004, 2005; Krishna et al. 2006). The step from any given PUF to the next higher free energy PUF involves the unfolding of one more foldon. This behavior describes a reversible sequential unfolding pathway that proceeds up a ladder of increasing stability, as in reaction Scheme 1, except for an optional branching at the initial red/infrared unfolding step. In Scheme 1, a one-letter color code specifies each foldon unit in Figure 1A according to its increasing unfolding free energy in the order infrared to red to yellow to green to blue. Each PUF is described by the foldons that are unfolded (lowercase) and still folded (uppercase).

Mallela M.G. Krishna, et al. Protein Sci. 2007 September;16(9):1946-1956.
4.
Figure 3.

Figure 3. From: Branching in the sequential folding pathway of cytochrome c .

(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 ΔGHX values, calculated from the logarithm of the measured HX rates, are accurate to better than 0.1 kcal/mol. (B) ΔGHX 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 ΔGHX values tend to merge into a single isotherm, reflecting a common unfolding reaction. (C) ΔGHX for amides in the green helix of oxidized WT Cyt c as f(pH). The green helix marker (Leu68) registers a smaller ΔGHX change with pH than the green loop. (D) ΔGHX 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.

Mallela M.G. Krishna, et al. Protein Sci. 2007 September;16(9):1946-1956.
5.
Figure 1.

Figure 1. From: Branching in the sequential folding pathway of cytochrome c .

(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 ΔGHX. 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 (Cm = 2.75 M GdmCl). The ΔGHX 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.

Mallela M.G. Krishna, et al. Protein Sci. 2007 September;16(9):1946-1956.
6.
Figure 2.

Figure 2. From: Branching in the sequential folding pathway of cytochrome c .

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 ΔGHX measured from the marker protons for each foldon. ΔΔGHX 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 Figure 3B. The value at pDr 7 represents a minimum estimate for the ΔGHX of the green loop unfolding (indicated by an upward arrow) since Leu32 exchanges more rapidly (with lower ΔGHX) 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 ΔGHX within 0.2 kcal/mol, but are shown vertically displaced in the figure for clarity.

Mallela M.G. Krishna, et al. Protein Sci. 2007 September;16(9):1946-1956.

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