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- Materials and Methods
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.
Abbreviations: Cyt c, cytochrome c; WT, wild-type Cyt c; pWT, pseudo-wild-type recombinant equine Cyt c (H26N, H33N); HX, hydrogen exchange; NHX, native state hydrogen exchange; PUF, partially unfolded form; GdmCl, guanidinium chloride; pDr, pH of D2O solution read by glass electrode.
It now appears that proteins are made up of small structural units, called foldons, that continually unfold and refold even under fully native conditions (Maity et al. 2005). Figure 1A illustrates the five foldon units that together form the structure of cytochrome c (Cyt c) (Bai et al. 1995; Krishna et al. 2003b). The foldons are shown color-coded in order of their increasing free energy for unfolding, from infrared and red up through blue, the unfolding free energy of which is equal to the global stability. The same concerted native-like foldon units are consistently seen in different kinds of experiments including hydrogen exchange (HX) pulse labeling done during kinetic folding (Roder et al. 1988; Krishna et al. 2003a, 2004b) and native state HX done under equilibrium (EX2) and kinetic (EX1) conditions (Bai et al. 1995; Bai and Englander 1996; Milne et al. 1999; Hoang et al. 2002; Krishna et al. 2003b, 2006; Maity et al. 2005).
Figure 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 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.
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The discovery of foldon units came from studies on the mechanism of protein folding. Spectroscopic methods widely used to observe protein folding in real time can only detect intermediates that transiently accumulate and even then provide almost no detailed structural information. The HX pulse labeling experiment (Krishna et al. 2003a) can outline the structure of intermediates and characterize their thermodynamic and kinetic properties, but this capability is also limited to intermediates that accumulate during kinetic folding. Native state HX (NHX) methods take advantage of the thermodynamic principle that proteins must unfold and refold continually, cycling through all of their higher free energy forms even under native conditions (Bai et al. 1995). Under favorable conditions, measured HX can become dominated by these partially unfolded forms (PUFs). NHX measurements can then define the structure and the equilibrium and kinetic properties of these intermediate forms, all at an amino-acid-resolved level, even though they are only infinitesimally populated and invisible to other methods. Results available for a number of proteins reveal a small number of discrete PUFs in which some of the foldon units of the native protein remain folded and others are unfolded. The PUFs represent the intermediates in each protein's folding pathway. It appears that the folding free energy landscape for each protein is well represented by a small number of predominant local minima populated by native-like PUFs rather than an undifferentiated amino-acid-level continuum.
Cyt c folding starts with the blue foldon unit, and then moves down the energy ladder, forming and successively putting into place its foldon building blocks to progressively assemble the target native protein (Roder et al. 1988; Bai et al. 1995; Xu et al. 1998; Milne et al. 1999; Hoang et al. 2002; Krishna et al. 2003a,b, 2006; Maity et al. 2004, 2005). Each sequentially formed PUF is seen to be constructed from the prior one by the addition of one more foldon unit. Other proteins appear to do the same (Chamberlain et al. 1996, 1999; Fuentes and Wand 1998a,b; Chamberlain and Marqusee 2000; Chu et al. 2002; Silverman and Harbury 2002; Takei et al. 2002; Yan et al. 2002, 2004; Feng et al. 2003, 2005; Cecconi et al. 2005). Results available point to a sequential stabilization mechanism in which prior native-like structure templates the addition of incoming complementary foldons in an order that is determined by the same interactions that join the foldons in the native protein (Xu et al. 1998; Rumbley et al. 2001; Englander et al. 2002; Maity et al. 2004, 2005; Krishna et al. 2006). Related results indicate that the normally occurring unfolding–refolding behavior of foldon units can participate in other functional processes in addition to folding (Hoang et al. 2003; Krishna et al. 2003b; Maity et al. 2006).
One wants to understand the structural determinants and the properties of protein foldons. The foldon units in the best worked out case of Cyt c are coincident with its secondary structural units or pairs thereof (Fig. 1A), indicating that they, and the folding pathways that they assemble, depend on the same factors that determine the native state. However, foldons may diverge somewhat from the exact secondary elements that compose the native protein because, in the PUFs where foldons are formed and characterized, some native interactions are absent and nonnative interactions that help to energy minimize the PUFs may be present (Feng et al. 2003, 2005). Also, experimental uncertainties may under- or overestimate foldon extent. Attempts have been made to develop algorithms that might predict foldons in known proteins (Bryngelson et al. 1995; Panchenko et al. 1996; Fischer and Marqusee 2000; Weinkam et al. 2005; Hilser et al. 2006).
The green foldon in Cyt c (Fig. 1A) seems anomalous. It consists of two segments, an Ω-loop (residues 19–36) and the middle 60s helix, that are distant in sequence and have minimal structural contact. Do these segments obligately unfold and refold as a single cooperative unit? The present work finds that the green Ω-loop segment is selectively sensitive to decreasing pH, which can be traced to some buried protonatable groups. We exploit this condition to consider the obligatory nature of the connection between the green helix and the green loop. Results show that each segment is able to unfold and refold independently of the other, leading to a branch point in the folding/unfolding pathway.
Materials and Methods
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- Materials and Methods
Commercially available WT equine Cyt c (type VI from Sigma Chemical Co.) was further purified when necessary by reverse phase HPLC (Rumbley et al. 2002). A recombinant modified Cyt c (H26N, H33N), called pseudo-wild type (pWT) and its mutants were expressed in a high yield Escherichia coli system and purified as described elsewhere (Rumbley et al. 2002). All other chemicals were as previously described (Krishna et al. 2006).
In equilibrium NHX experiments, Cyt c was placed into D2O under mildly destabilizing but still strongly native conditions with low concentrations of denaturant or at reduced pH. Time-dependent H to D exchange at the various amide sites was measured by recording sequential 1H-1H COSY spectra (500 MHz Varian Inova with cold probe). From the measured HX rates the free energy of the opening reaction that exposes each hydrogen to exchange (ΔGHX) can be computed (Krishna et al. 2004b).
ΔGHX was calculated from the equation, ΔGHX = −RT ln Kop = −RT ln (kex/kch), which holds for Cyt c in the EX2 region below pH 10 (Krishna et al. 2004b). Here, kex is the measured exchange rate and kch is the chemical exchange rate calculated for the unprotected amide (Bai et al. 1993; Connelly et al. 1993). HX rates in well-defined cases are accurate to about 10%. ΔGHX calculated from the logarithm of HX rate is therefore accurate to better than 0.1 kcal/mol.
Typical protein concentration was ∼6 mM. Experiments were done at 20°C (unless otherwise specified) with appropriate pH buffers (0.1 M) and 0.5 M KCl to minimize charge effects on stability and HX. HX experiments and data analysis were done as described before (Krishna et al. 2004a). Dead time from the start of the HX reaction was ∼15 min. To collect faster time points, short gradient COSY spectra (2 scans, 23 min) were collected initially back to back.