Cytochrome c Folding Landscapes

4.2.1 Zinc-substituted Cytochrome c

The folding energy landscape of cytochrome c has been investigated by exploiting the widely different ET reactivities of buried and exposed zinc-substituted hemes [43]. Zn-cyt c is structurally homologous to the iron protein, which has been studied in great detail [94,129,136,158]. A nonnative axial ligand (His26 or His33) replaces Met80 at neutral pH in denatured equine Fe-cyt c [121]; the rate-limiting folding step in this case is correction of heme misligation. Owing to weaker binding and faster substitution at the sixth coordination site, replacement of Fe with Zn will eliminate axial ligand traps during refolding [159].

Addition of GuHCl to solutions of Zn-cyt c produces a blue shift in the Soret absorption band [43], giving a species with a spectrum similar to that of Zn(II)-substituted myoglobin [160]. GuHCl unfolding curves generated from UV-vis absorption and far-UV CD spectra show that the stability of folded Zn-cyt c is comparable to that of the Fe(III) form [36,161]. In contrast to Fe-cyt c, the zinc center in the unfolded protein is not coordinated by any peptide side chain other than the native His18 [159].

Changes in Zn-cyt c Soret absorption after stopped-flow dilution of denaturant were examined: the transient absorption kinetics are exponential functions and the observed rate constants depend linearly on denaturant concentrations. The extrapolated time constant for refolding in the absence of denaturant is about a millisecond. The Zn-cyt c folding rate is about 10 times higher than that of the Fe(III) protein at comparable driving forces [94,129], consistent with the absence of heme misligation.

More complex Zn-cyt c folding becomes apparent when the process is probed with transient absorption measurements of *Zn-cyt c/Ru(NH3)3+ ET kinetics. As the polypeptide chain folds around the porphyrin, the *Zn-cyt c ET rate decreases from its value in the unfolded protein (~7 x 106 s-1,[Ru(NH3)3+] = 3-5 mM) to that characteristic of folded molecules (~2.5 x 105s-1). Biphasic *Zn-cyt c decay kinetics are observed 1 ms after GuHCl dilution: two-thirds of the excited Zn-porphyrins decay in a fast phase (6.8 x 106 s-1) attributable to unfolded protein; the remaining third exhibits a rate constant (4.1 x 105s-1) closer to that expected for folded molecules. ET kinetics measured at longer folding times remain biphasic, with the amplitude of the faster component decreasing in favor of an increase in the amplitude of the slow component (Figure 13 upper panel). Both *Zn - cyt c decay rates decrease by about a factor of 2 (1ms: 6.8 x 106,4.1 x 105 s-1; 50 ms: 3.2 x 106, 2.4 x 105s-1) as the folding reaction proceeds (Figure 13, upper panel). This variation in quenching efficiency reflects a gradual collapse of polypeptide structures during folding, revealing the conversion of compact, nonnative conformations to a fully folded protein.

The amplitudes of the two *Zn-cyt c decay phases vary exponentially with folding time and the rate constant is in reasonable agreement with that measured by Soret absorption spectroscopy (Figure 13, lower panel). It is noteworthy that, at the earliest measured folding times (1 ms), there are significant amplitudes in both the fast and slow *Zn-cyt c ET phases. This is substantially more than would be expected on the basis of the stopped-flow dead-time (~1ms) and the observed rate constants extracted from the signal amplitudes. Measurements of *Zn-cyt c kinetics at different GuHCl concentrations consistently extrapolate back to a burst-phase ensemble [94,128,129,162] with a 2:1 ratio of fast and slow ET components (Figure 13, lower panel): these results demonstrate that the burst ensemble is heterogeneous; molecules in one-third of the protein population have compact structures, and ones in the remaining fraction have exposed Zn-porphyrins.

It is apparent that there is underlying complexity in Zn-cyt c folding. The fraction of the burst ensemble with slow *Zn-cyt c decay kinetics could be fully folded protein or an ensemble of compact nonnative structures. The former possibility would be an example of fast-track folding [163-165], where about a third of the unfolded Zn-cyt c molecules adopt conformations that can

A + 0 ms

I ■


A + 1 ms

A + 2 ms


A + 3 ms



■ ■


A + 50 ms


time, ms

Figure 13. Zn-cyt c folding probed by ET kinetics. Upper panel: Bimolecular quenching rate constants and relative amplitudes from biexponential fits of *Zn-cyt c during folding ([Zn-cyt c] = 10 |xM, [Ru(NH3)6+] = 4mM, [GuHCl]final = 1.46M, pH 7,A is the stopped-flow mixer dead-time ~1ms). Lower panel: Amplitudes of fast and slow *Zn-cyt c decay constants as functions of time after the initiation of protein folding. The amplitudes vary exponentially (kfold = 33 s-1) with folding time and extrapolate to a ~2:1 fast:slow ratio at time zero ([GuHCl]final = 1.46M).

time, ms

Figure 13. Zn-cyt c folding probed by ET kinetics. Upper panel: Bimolecular quenching rate constants and relative amplitudes from biexponential fits of *Zn-cyt c during folding ([Zn-cyt c] = 10 |xM, [Ru(NH3)6+] = 4mM, [GuHCl]final = 1.46M, pH 7,A is the stopped-flow mixer dead-time ~1ms). Lower panel: Amplitudes of fast and slow *Zn-cyt c decay constants as functions of time after the initiation of protein folding. The amplitudes vary exponentially (kfold = 33 s-1) with folding time and extrapolate to a ~2:1 fast:slow ratio at time zero ([GuHCl]final = 1.46M).

refold very quickly. The remaining protein molecules have relatively exposed porphryin groups, and fold on a substantially longer time scale. Alternatively, the fast and slow *Zn-cyt c decay components formed immediately after dilution of [GuHCl] may reflect a shift in the equilibrium between unfolded and compact nonnative structures. Although it is often assumed that native solvent conditions will strongly favor compact structures, the *Zn-cyt c ET kinetics clearly indicate that two-thirds of the molecules in the burst intermediates have highly exposed porphyrins. Ultimately, the entire protein ensemble folds because, at low [GuHCl], the native structure is much more stable than unfolded conformations.

4.2.2 AEDANS-labeled Cytochrome c

In our laboratory, Lyubovitsky examined DA distance distributions during the folding of AEDANS-labeled S. cerevisiae iso-1 cytochrome c [48]. It was shown in prior work that the folding kinetics of iso-1 cyt c are comparable to those of the more extensively studied equine protein [36,94,166-169]. In the folded protein at neutral pH, an imidazole nitrogen (His18) and a thioether sulfur (Met80) axially ligate the heme iron (Figure 8). The nitrogenous base of an amino acid side chain (pH 7: His26, His33, His39) replaces Met80 in the unfolded protein [121]. This misligation retards refolding, because ligand exchange is required for the peptide to adopt its native conformation [40,170].

The AEDANS fluorescence intensity, a measure of the ensemble-averaged extent of folding in AEDANS(C102)-cyt c, exhibits a biphasic kinetics profile when the refolding is triggered by stopped-flow dilution of GuHCl. The major fraction (90%) of the decrease in fluorescence occurs within 2 s of mixing; the remaining 10% of the signal change proceeds in the 2-10 s time interval [48]. The integrated AEDANS fluorescence only provides an indication of the extent of folding and reveals nothing about the polypeptide ensemble heterogeneity.

Measurements of I(t) at various times during folding (Figure 14) provide snapshots of DA distance distributions, P(r). Immediately after the folding is triggered (1 ms), 40% of the protein ensemble has collapsed, producing a population with

Figure 14. AEDANS fluorescence decay kinetics measured at the indicated times after initiation of AEDANS(C102)-cyt c refolding.

Figure 14. AEDANS fluorescence decay kinetics measured at the indicated times after initiation of AEDANS(C102)-cyt c refolding.

1 ms

—r""f r M ' i'—H

10 ms .in, . i I

i i i i i i

40 ms


380 ms li.


760 ms

16 s 16 s

Figure 15. Evolution of the distributions of DA distances (P(r)) during the refolding of AEDANS(C102)-cyt c ([GuHCl]final = 0.13 M, pH 7).

an average DA distance of ~27Â (Figure 15). Surprisingly, 60% of the protein remains in extended conformations with DA distances greater than 40 Â. Within 10 ms, the P(r) distribution develops a component at r = 25 Â, a value comparable to that of the folded protein. By 50 ms, the 25-Â component is larger than the 27-Â population, and after 400 ms, the latter fraction has nearly disappeared (Figure 15). Concomitant with the evolution of the collapsed ensemble, there is a decrease in the population of extended conformations.

The formation of correctly folded cyt c is limited by heme axial ligand substitution processes at neutral pH [170]. Displacement of misligated His groups in denatured cyt c by imidazole dramatically accelerates refolding [30]. NMR investigations of the imidazole adduct of equine cyt c reveal only modest disruption of the protein structure in the vicinity of the Met80 residue [171]. Measurements of FET kinetics during AEDANS(C102)-cyt c refolding in the presence of imi-dazole at room temperature indicate that the native DA distance distribution is formed in less than 20 ms. At lower temperature (1 °C), the evolution of DA distance distributions is remarkably similar to that in the absence of imidazole. The key difference is the overall time scale of refolding; formation of folded protein is largely complete within 200 ms. Roughly equal populations of extended (r > 50 Á) and collapsed (r = 32 Á) structures are observed immediately after folding is triggered. The 32-Á distribution evolves into a 25-Á native distribution in ~200 ms.

The FET kinetics measured during AEDANS(C102)-cyt c folding indicate that dilution of denaturant to concentrations favoring native protein conformations does not produce a complete collapse of the polypeptide ensemble. Within the deadtime of stopped-flow measurements, only half of the protein population has formed compact structures. The compact ensemble (C) is characterized by a mean AEDANS-heme separation of ~27Á. This distance is greater than that of the native protein, indicating that the collapsed species are not fully folded. As the population of proteins with the native fold (N) increases, the extended (E) and compact (C) populations disappear on comparable time scales.

It is surprising that such a large fraction of the protein ensemble remains in an extended conformation after denaturant dilution. These extended conformations are not a consequence of His misligation in the unfolded protein; high concentrations of imidazole displace the His ligands and speed refolding, yet both E and C fractions are observed at 1 °C. The accelerated cyt c refolding in the presence of imidazole also demonstrates that E does not arise from kinet-ically trapped conformations containing proline isomers or incorrect topomers; there is no obvious mechanism by which added imidazole could eliminate such traps. Indeed, the parallel disappearance of E and C, rapidly in the presence of imidazole, slowly when His residues misligate the heme, strongly suggests that the two populations are in rapid equilibrium [129,169].

The time-resolved DA distance distributions extracted from FET measurements lead to an idealized folding landscape for DNS(C102)-cyt c (Figure 16). The lateral dimension of the landscape is the deviation of the DA distance from its value in the folded protein (R - RF;RF ~ 25 Á); and the vertical axis reflects the polypeptide free energy. The cross-section of this landscape (Figure 16, right panel) illustrates two possible fates for a polypeptide that was in an extended conformation (r > 40 Á) prior to the initiation of folding. Denaturant dilution shifts the E ^ C equilibrium to produce comparable populations of extended and compact polypeptides undergoing rapid exchange (~100 ^s). Collapsed conformations (C) with favorable arrangements of the polypeptide backbone can transform into the native structure (N) at pH 7 by surmounting an energy barrier corresponding to a heme axial-ligand substitution process (Figure 16, right panel, left path). Rapid collapse of a polypeptide is likely to produce conformations that cannot evolve into the native fold (C^N) because of topological frustration [164] (Figure 16, right panel, right path). This model implies that the population of collapsed polypeptides is heterogeneous and that only a fraction of the collapsed conformers is competent to transform into N [164,172].

Figure 16. Idealized representations of the AEDANS-labeled cytochrome c folding landscape. The lateral dimension in both plots is the deviation from the native DA distance (~25A). The three-dimensional folding landscape (left panel) reveals a flat region on the periphery for extended and compact polypeptides that surrounds the global energy minimum (blue) of the native (N) fold. Some of the compact structures are separated from the native fold by low energy barriers (yellow). The remainder have high barriers (red) to native state formation. Polypeptides that fall into these minima must rearrange to extended conformations that can collapse into productive compact structures. The two-dimensional cross-section of the landscape (rightpanel) shows nearly degenerate, shallow energy minima corresponding to extended (E) and collapsed (C, C') conformations. The collapsed structures on the left side (C) of the global energy minimum can surmount a ligand substitution barrier (blue curve) which can be lowered by the addition of imidazole (red curve) to reach the native (N) structure. Collapsed peptides on the right side (C') face a high barrier to formation of the native fold; this population must exchange with other collapsed structures with lower barriers to folding.

For topologically frustrated compact conformations, the only route to the native state involves formation of an extended polypeptide that can recollapse to a more favorable structure. This mechanism is illustrated by the three-dimensional contour plot of the AEDANS(C102)-cyt c folding landscape (Figure 16, left panel). The native fold is represented by the central free-energy minimum. Owing to the near degeneracy of collapsed and extended polypeptides, the landscape consists of a relatively flat outer rim surrounding a narrow funnel. Rapid interchange among extended conformations via intrachain diffusion proceeds on the outer rim of the landscape [136,139,173]. These extended polypeptides frequently fall into collapsed conformations toward the interior of the landscape; some of these (4 of the 12 collapsed conformers shown on the idealized surface (Figure 16, left panel)) can form the native structure; the others must extend and try again.

Fe-cyt c + imidazole/.!



Collapsed and extended polypeptides in rapid equilibrium at the top of the funnel must wait for a ligand substitution event to open the way to conversion to the native structure at the bottom. Addition of imidazole lowers the ligand substitution barrier and speeds the transformation of collapsed polypeptides into the native form.

The picture that emerges is one in which extended polypeptide conformations play a pivotal role in AEDANS(C102)-cyt c refolding. Time-resolved FET measurements reveal that, at the onset of folding, fully half of the polypep-tide ensemble is found in extended conformations reminiscent of the denatured protein [128,174]. The near degeneracy and rapid equilibration of collapsed and extended populations would enable AEDANS(C102)-cyt c to escape from topologically frustrated compact structures that cannot produce the native fold because of extremely high energy barriers [164]. If collapsed intermediates were substantially more stable than extended geometries, formation of extended structures would occur infrequently, exacerbating the problem of topological frustration [164]. Instead, AEDANS(C102)-cyt c can collapse and extend many times as it searches for compact structures that have low-barrier routes to the native conformation [172].

Since collapsed cyt c intermediates are not substantially more stable than the fully denatured protein, the likelihood of a native structure rearranging to a partially folded species is substantially lower than would have been the case if collapsed conformations were found in deeper wells on the folding landscape. If this is a common protein folding characteristic, it may be an important means of avoiding the partially folded intermediates that can aggregate into the misfolded structures that characterize a variety of disease states.

4.2.3 Cobalt-substituted Cytochrome c

Years of experimental work on the energetics and dynamics of self-assembly of cytochrome c have failed to resolve whether unfolded polypeptides undergo global collapse to compact conformations upon dilution or laser triggering to solution conditions that strongly favor the native structure [30,35,94,129,130,158,166,169]. Indeed, recent kinetics experiments suggest that comparable populations of compact and extended polypeptides are formed rapidly (< 1ms), and these two populations disappear in parallel as the protein folds [43,48]. To test this new energy landscape model rigorously, Tezcan isolated the structures that comprise the burst-phase by replacing the native iron center with cobalt(III) [175]: owing to very high ligand substitution barriers, the final step of cobalt(III)-cytochrome c (Co-cyt c) folding is well separated in time from all early events; and, in this kinetically stabilized system, the early folding intermediates can be examined carefully over a period of hours by standard physical methods [52].

The folding free energies (AGf) of equine, tuna, and yeast Co-cyt c (9.9, 9.8, and 6.1kcal/mol, respectively) determined by monitoring changes in far-UV CD and Soret absorption spectra in GuHCl solutions are comparable to those of the corresponding Fe(III)-protein (9.0, 9.4, and 4.6kcal/mol) [36,161]. The folding thermodynamics accord with the crystal structure of tuna Co-cyt c (1.5-Á resolution), which confirms that replacing Fe(III) with Co(III) does not disrupt the native protein fold (0.22-Á RMS Ca displacements in Fe(III)- and Co(III)-proteins) [176,177].

Both equine and tuna Co-cyt c refold several orders of magnitude more slowly than the corresponding native protein; the observed kinetics are biphasic, owing to the presence of misligated unfolded protein populations [175] with different activation energies for ligand substitution. In the equine protein, His26 and His33 are the likely axial ligands; and there is evidence that Lys side chains coordinate to Co(III) in the unfolded tuna protein, which lacks His33 [175].

Analysis of the equine Co-cyt c folding kinetics suggests that His33 binds more tightly to the metal center than His26 in the unfolded protein [175]. The refolding phases associated with His26- and His33-misligated proteins exhibit comparable amplitudes after relatively short incubation periods in denaturant solution, indicating that the binding rates for the two histidines are similar [175]. However, with longer incubation time, the dissociation rate for His26 is 3 times greater than that for His33, a finding that is in excellent agreement with the 3- to 4-fold faster folding rate for the His26-misligated protein. It is apparent from these results that folding is limited by His dissociation from Co(III); and that the weaker binding of His26 to the metal center (relative to His33) is due to unfavorable steric interactions and the lower entropy associated with formation of a smaller loop [121].

Pulsed H/D exchange NMR measurements have revealed cyt c folding intermediates in which the C- and N-terminal helices are docked [30,178]. As expected, then, there is a large decrease in the far-UV CD amplitude (40% of total amplitude) immediately after diluting equine Co-cyt c/GuHCl solutions (Figure 17). The helical intermediate persists at low temperature for several hours without significant change. The CD spectrum of the Co-cyt c folding intermediate is similar to that reported recently for its Fe-protein analogue [174].

Unfolded Co-cyt c displays a 1H NMR spectrum characteristic of a random coil: the resonances are sharp and not dispersed. The upfield signals attributable to Met80 side chain protons [179] in the spectrum of the folded protein are absent, consistent with His-misligation. Shortly after the initiation of folding, when helical intermediates are fully populated, resonances in the NMR spectrum are broadened and overlapped, but remain negligibly dispersed. Such a spectrum indicates conformational exchange times comparable to the chemical shift time scale (~ ms). Thus, the NMR experiments confirm that folding intermediates containing elements of secondary structure are in rapid equilibrium with unstructured conformations.

210 220 230 240 250

210 220 230 240 250

Figure 17. Far-UV CD spectra of equine Co-cyt c species observed during refolding in 0.4 M GuHCl at 7°C: unfolded (circle), t ~ 1, 10, 55min, and folded (triangle).

FET kinetics provide nanosecond-time-scale snapshots of DA distance distributions in rapidly equilibrating populations of folding intermediates [48]. FET kinetics from AEDANS at Cys102 to the Co(III)-porphyrin were monitored during yeast Co-cyt c folding. Similar to the iron protein [48], AEDANS(C102) fluoresces intensely when unfolded by GuHCl; and it is quenched by the Co-porphyrin in the folded structure. As expected, the folded protein has a narrow DA distance distribution centered at ~27A and, notably, the unfolding process is accompanied by a broadening of P(r) with appearance of extended conformations with r > 50 A.

A fraction of the AEDANS(C102)-Co-cyt c population collapses shortly after triggering folding by manual dilution of denaturant (Figure 18) [52]. Interestingly, however, most of the molecules are still in extended conformations hours after initiating the refolding reaction; and, on an even longer time scale, both extended and compact conformations disappear as native structure forms. The time required to reach P(r) of the Co-cyt c is >18h.

Comparison of the FET kinetics for Co-cyt c with those obtained for Fe-cyt c [48] is especially revealing. Immediately after triggering AEDANS(C102)-Fe-cyt c folding (~1ms), the measurements reveal a bimodal distribution of AEDANS-Fe(III) distances: half of the protein molecules adopt compact unfolded plHln..l| I J

3 h 40 min

12 h

17 h 45 min folded

Figure 18. Evolution of the distributions of DA distances (P(r)) during the refolding of AEDANS(C102)-Co-cyt c ([GuHCl]final < 0.5M,pH 7).

structures and the remainder are in extended conformations. As folding progresses, the compact and extended populations decrease in parallel and are replaced by folded protein. It is apparent, then, that similarly structured intermediates are populated in both the Fe(III) and Co(III) proteins. The key difference between the two is the refolding time scale: Fe-cyt c evolves to the native structure in a few hundred milliseconds, whereas folding the Co(III)-protein requires several hours.

The combined spectroscopic probes of the burst-phase folding ensemble in Co-cyt c shed new light on the nature of polypeptide structures that form immediately after dilution of denaturant solutions. CD spectra demonstrate that there are elements in the ensemble with some secondary structure. Since the FET kinetics reveal populations of both compact and extended structures, it is likely that the compact structures give rise to the CD signal. The absence of dispersion in the NMR spectra demonstrates that the compact and extended structures equilibrate on a submillisecond time scale. The Co-cyt c folding data also are consistent with time-resolved small-angle X-ray scattering measurements that show that the Fe-cyt c ensemble has a bimodal pair distribution function a few milliseconds after the initiation of folding [128].

The possibility that global hydrophobic collapse is not an obligatory step in protein folding has been examined in computational experiments [18]. Relative stabilities of collapsed and extended conformations were suggested to correlate with the properties of the primary sequences and overall stability of the folded protein: more hydrophobic and less optimized sequences favored formation of collapsed intermediates, whereas extended structures evolved directly to the native fold in less hydrophobic and strongly optimized sequences [18]. The sequence and stability of cyt c appear to place it between these two limits: in this case, extended and collapsed structures are degenerate; and nearly all of the folding free energy is released when the compact conformations convert to the native fold. The relative instability of collapsed nonnative structures not only prevents formation of misfolded structures during the self-assembly process, but also reduces the probability that the native protein will transiently adopt an incorrect conformation.

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