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1 Chemistry Department, Tulane University, New Orleans, Louisiana 70118, USA
2 Molecular and Cellular Biology Graduate Program, Tulane University, New Orleans, Louisiana 70118, USA
3 Instituto de Technologia Quimica e Biologica, Universidade Nova de Lisboa, 2780-156 Oeiras, Portugal
Reprint requests to: Pernilla Wittung-Stafshede, Chemistry Department, Tulane University, 6823 St. Charles Avenue, New Orleans, LA 70118-5698, USA; e-mail: pernilla{at}tulane.edu; fax: (504)-865-5596.
(RECEIVED November 29, 2000; FINAL REVISION April 27, 2001; ACCEPTED May 3, 2001)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.49401
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H[Tm]) and Tm from which an upper limit for the heat capacity of unfolding (CP) was determined to be 3.15 ± 0.1 kJ/(mole • K). Only by the use of the stronger denaturant guanidine thiocyanate (GuSCN) is unfolding of A. ambivalens ferredoxin at pH 7 (20°C) observed ([GuSCN]1/2 = 3.1 M; GU[H2O] = 79 ± 8 kJ/mole). The protein is, however, less stable at low pH: At pH 2.5, Tm is 64 ± 1°C, and GuHCl-induced unfolding shows a midpoint at 2.3 M (GU[H2O] = 20 ± 1 kJ/mole). These results support that electrostatic interactions contribute significantly to the stability. Analysis of the three-dimensional molecular model of the protein shows that there are several possible ion pairs on the surface. In addition, ferredoxin incorporates two iron–sulfur clusters and a zinc ion that all coordinate deprotonated side chains. The zinc remains bound in the unfolded state whereas the iron–sulfur clusters transiently form linear three-iron species (in pH range 2.5 to 10), which are associated with the unfolded polypeptide, before their complete degradation. Keywords: Hyperthermophiles; thermostability; iron-sulfur proteins; protein unfolding
Abbreviations: GU, unfolding free energy • Tm, melting temperature • Fd, ferredoxin • GuHCl, guanidine hydrochloride • H[Tm], enthalpy of unfolding • CP, heat capacity of unfolding • GuSCN, guanidine thiocyanate • CD, circular dichroism
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Three models have been proposed to explain the higher denaturation temperatures of thermophilic proteins (Rees and Adams 1995; Beadle et al. 1999); each has a different thermodynamic consequence. In the first model, compared with a mesophilic protein, the thermophilic one could be more thermodynamically stable throughout the temperature range, that is, have higher free energy of unfolding (GU) than the mesophilic protein at every temperature. A second model predicts that the free energy profile of the thermophilic protein will be displaced horizontally to a higher temperature. In this model the maximal value for GU would be equal for the mesophilic and thermophilic protein, but the maxima would occur at different temperatures. At high temperatures, the thermophilic protein would be more stable; at lower temperatures, the mesophilic protein would be more stable. Finally, a third model predicts that the free energy profile for the thermophilic protein would be a flattened version of that for the mesophilic protein. Thus, the thermophilic protein would have a more shallow dependence of GU on temperature, but the maximal GU would again be equal for the mesophilic and thermophilic proteins. Support for the different models, as well as combinations thereof, have been reported (McCrary et al. 1996; Beadle et al. 1999; Hollien and Marqusee 1999).
To gain better insight into mechanisms governing protein thermostability, simple models may be of great use. Ferredoxins are good examples of such proteins: They are small, monomeric polypeptides containing iron–sulfur centers whose integrity can be followed easily by spectroscopic methods. In addition, ferredoxins are widespread in the three domains of life; they are evolutionarily ancient proteins considered to have been the first bioinorganic catalysts (Otaka and Ooi 1987; Wachtershauser 1992). Because archaea are presumed to be the most ancient living organisms on contemporary Earth (Woese et al. 1990), the study of ferredoxins from such thermophilic organisms may elicit ancestral strategies for protein stabilization. Moreover, it may help to decide whether life originated at high or low temperatures (Pace 1991). A family of di-clusters, seven-iron-containing ferredoxins from various archaea belonging to the order Sulfolobales, has been characterized (Teixeira et al. 1995; Gomes et al. 1998). These organisms live optimally at pH 2–4.5 and temperatures of 65°C–80°C. Their ferredoxins are acidic, 
12 kD proteins that contain one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ center. The structure of the ferredoxin from Sulfolobus strain 7 (Fujii et al. 1997) revealed the presence of a zinc center, in addition to the iron clusters, in this protein. By monitoring the alteration of the spectral properties as a function of time at a high temperature, the folded forms of the Sulfolobus ferredoxins were shown to be extremely resistant to degradation (Teixeira et al. 1995; Gomes et al. 1998). We recently reported a preliminary study (Wittung-Stafshede et al. 2000) of the stability and unfolding of the Acidianus ambivalens ferredoxin, for which the complete amino acid sequence and a structural model are available, that revealed that this ferredoxin was highly resistant to both temperature (Tm of 122°C; pH 7) and chemical perturbation (addition of 8 M guanidine hydrochloride [GuHCl] did not unfold the ferredoxin at pH 7, 20°C). At pH 10, GuHCl-induced ferredoxin unfolding occurred with a midpoint at 6.3 M, and a GU in water (pH 10; 20°C) of 70 ± 3 kJ/mole was reported (Wittung-Stafshede et al. 2000).
In the current investigation, we explore further the thermostability of the A. ambivalens ferredoxin (Fd). We show that the stability of Fd is extremely high, having a GU near 80 kJ/mole at pH 7, 20°C. From thermal unfolding experiments a low heat capacity of unfolding (CP) is estimated, indicating a rather flat stability versus temperature profile. Lowering the pH decreases the stability dramatically (both GU at 20 °C and Tm), indicating that electrostatic interactions contribute favorably to the high stability of Fd at neutral pH. Upon polypeptide unfolding, at both high and low pH, the iron–sulfur clusters first rearrange into intermediate (potentially linear three-iron) species before dissociation and decomposition occur. Interestingly, the zinc ion remains coordinated to the unfolded protein.
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). The reported experiments are all performed with Fd in its fully oxidized form (the resting state); native oxidized Fd displays distinct visible absorption at 410 nm (
410 = 30,400/M per cm; see Fig. 1B). The melting temperature (Tm) for Fd in buffer (pH 7) was determined from temperature-induced unfolding experiments monitored by far-UV CD in the presence of various amounts of GuHCl, because the presence of GuHCl lowers the melting temperature. The Tms found in the presence of different GuHCl concentrations were used earlier to estimate, by linear extrapolation, a Tm of 122 ± 2°C for Fd at pH 7 in the absence of denaturant (Wittung-Stafshede et al. 2000). We now performed additional thermal unfolding experiments and analyzed all melting transitions according to a modified van't Hoff equation (see Materials and Methods).
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H values were identical, and CP from calorimetric and H versus Tm experiments agreed, the investigators argued that equilibrium thermodynamics had been obtained (Pfeil et al. 1997). It should be noted, however, that there was no measure of the possible enthalpy changes associated with cluster dissociation and decomposition in the T. maritima study; in addition, we have found no such estimates anywhere in the literature. Nevertheless, because the unfolding processes are likely to be similar for the two ferredoxins, and experimental results on A. ambivalens Fd add direct support, a thermodynamic treatment of our data appears valid as an approximation.
Table 1 lists the enthalpy of unfolding (H[Tm]) values that were derived at the different Tms. A linear relationship between H(Tm) and Tm was found (Fig. 2), indicating that CP is independent of temperature; a linear fit provides a CP value of 3.15 ± 0.1 kJ/(mole • K). No correction of the H values at pH 7 for possible (in the presence of high GuHCl concentrations) contributions of GuHCl–protein interactions were made. However, CP truly equals CP(protein unfolding) + CP(GuHCl interactions), so that the experimentally determined CP value is an upper limit of CP for protein unfolding (Makhatadze and Privalov 1992; Agashe and Udgaonkar 1995). Because the H values for A. ambivalens Fd at pH 2.5 (at no, or low, GuHCl concentration) and at pH 7 correlate (Fig. 2), contributions to H from GuHCl–protein interactions appear small. Moreover, potential errors in the H values because of irreversibility are assumed negligible. If the H values are affected by the reactions leading to irreversibility, the effect would be similar at all temperatures and, therefore, the slope of H versus T (i.e., CP) would not be perturbed (see Discussion). Strikingly, at pH 2.5 conditions, thermal unfolding takes place at an almost 60°C lower temperature than at pH 7: Tm is 64°C and 122°C in buffer at pH 2.5 and pH 7.0, respectively (Table 1).
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CP and H[Tm] by a modified Gibbs-Helmholtz equation [Privalov et al. 1986; McCrary et al. 1996]), whereas at this temperature and pH 2.5 conditions the native state of Fd is not favored thermodynamically (Tm[pH 2.5] < 80°C). Although the organism grows optimally at low pH, the interiors of the cells do not necessarily adopt acidic pH. Indeed, for the closely related archaeon Thermoplasma acidophilum, also growing at pH 2, an internal pH of 6.6 has been measured (Hsung and Haug 1975).
Chemically induced Fd unfolding at neutral pH
A concentration of 8 M GuHCl did not promote Fd to unfold fully at pH 7 (Wittung-Stafshede et al. 2000), and no GU(H2O) at 20°C, pH 7 has been reported. We now used guanidine thiocyanate (GuSCN; Cota and Clarke 2000), which is a stronger denaturant than GuHCl, to promote Fd unfolding at pH 7. GuSCN absorbs far-UV light to a significant extent, prohibiting the use of the change in Fd's far-UV CD signal to monitor GuSCN-induced unfolding. Instead, Fd unfolding was monitored by changes in Trp emission; upon protein unfolding the fluorescence increases in intensity, and the maximum shifts from 345 ± 2 to 355 ± 3 nm (Fig. 1C
). An identical equilibrium transition was observed by monitoring the disappearance of visible absorption (data not shown). Moreover, GuHCl-promoted unfolding of Fd at lower pHs (discussed below) revealed that, at each pH, identical transition midpoints and GUs were found, independent of whether far-UV CD, emission maximum, or visible absorption was monitored. Also, Fd samples at conditions near the unfolding midpoints could be incubated for up to 24 h without changes in their spectroscopic properties. Taken together, these results support that equilibrium unfolding of Fd can be treated as an apparent two-state process and that estimations of thermodynamic values are appropriate.
Titration of Fd with GuSCN (pH 7) yields a sharp transition with a midpoint at 3.1 M GuSCN, shown in Figure 3. Using a two-state fit, GU(H2O) at pH 7 (20°C) was calculated to be 79 ± 8 kJ/mole. Comparing GuHCl- and GuSCN-induced unfolding experiments with myoglobin provided a scaling factor of 2.3 for interconverting between GuSCN and GuHCl unfolding midpoint concentrations (an identical factor was derived from data in Cota and Clark [2000]). Using this scaling factor, the apparent midpoint for a GuHCl titration of Fd at pH 7 would occur at 7.1 M GuHCl.
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). For many proteins, as also appears to be the case for the Fd, the highest thermodynamic stability is found around pH 7, regardless of whether the isoelectric point of the protein is acidic or basic (the A. ambivalens Fd is acidic). To characterize the pH dependence for this Fd thermodynamic stability further, GuHCl titrations were performed at pH 2.5, 4.0, 5.2, 8.5, and 10. The transitions were monitored by far-UV CD (220 nm), visible absorption (410 nm), and tryptophan emission (350 nm); all methods gave identical results for each condition. The transition midpoints and corresponding GU(H2O) are listed in Table 2 and shown in Figure 4. It is clear from the data that there is a strong pH dependence of the Fd stability. The stability decreases drastically below pH 5, although it appears almost constant above pH 7 (only slightly lower at pH 10 than at pH 7). At pH 2.5, GuHCl-induced unfolding occurs with a midpoint of 2.3 M GuHCl, and a GU(H2O) of 20 ± 1 kJ/mole was estimated (which is less than a third of the stability at pH 7).
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To address if transient linear [3Fe-4S] intermediates (not detected in the equilibrium experiments) are also observed upon Fd unfolding at lower pH, we performed additional kinetic studies. GuHCl-induced unfolding kinetics of Fd at pH 2.5, using stopped-flow mixing because of the rapid reaction (Fig. 5A), reveals the transient appearance of absorption at 610 nm (1/ku = 1.7 ± 0.1 sec, 4 M GuHCl, pH 2.5). This phase is followed by a slower decrease to zero absorption intensity (1/ku = 120 ± 5 sec, 4 M GuHCl, pH 2.5). Moreover, on adding 3.5 M GuSCN to Fd at pH 7, a 610-nm absorption band appears within the first minute after mixing. This process is followed by a slower (over several minutes) decrease, resulting in complete disappearance of visible absorption at both 410 and 610 nm. In identical pH 7 conditions, 520- and 610-nm absorption bands are also observed transiently with the Sulfolobus acidocaldarius seven-iron ferredoxin (Fig. 5B). The S. acidocaldarius ferredoxin shares 89% amino acid identity with the A. ambivalens Fd. Thus, in a wide pH range (2.5 to 10), the unfolding path for the A. ambivalens Fd appears to involve a transient state in which the polypeptide coordinates rearranged, possibly linear [3Fe-4S], iron clusters.
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GU(H2O) values (investigated at pH 2.5 and pH 7; data not shown), (4) identical transition midpoints and GU(H2O) values when unfolding is monitored by different spectroscopic probes, and (5) protein incubation near unfolding midpoints, for up to 24 h, does not result in spectroscopic changes. It is not known if the irreversible steps during Fd unfolding, believed to be iron–sulfur cluster dissociation and decomposition, contribute to the estimated enthalpy changes. This causes concern with respect to the validity of the absolute H values. However, because an enthalpic effect from the irreversible steps should be similar at each Tm, the slope of the H versus Tm plot will still give a reliable CP value.
Fd stability as a function of temperature
It is clear that A. ambivalens Fd is very stable toward both thermal and chemical denaturant unfolding near neutral pH. At pH 7 (20°C), we estimated the GU for Fd to be 79 ± 8 kJ/mole. This high thermodynamic stability is in accord with the high thermal melting point estimated at pH 7 (Tm = 122°C). Ferredoxin from T. maritima (60 residues, one [4Fe-4S] cluster) was also reported to be stable beyond the boiling point of water (Pfeil et al. 1997). CP relates to the amount of hydrophobic surface area exposed upon unfolding. The estimated CP of 3.15 ± 0.1 kJ/(mole • K) for the A. ambivalens Fd, determined from thermal melts at different buffer/denaturant conditions (Fig. 2; Table 1), is low. Data sets for a large number of mesophilic proteins have yielded an average value for CP of 59 J/(mole • K) per residue (Makhatadze and Privalov 1995; Myers et al. 1995; Robertson and Murphy 1997); this corresponds to a predicted CP of 6.1 kJ/(mole • K) for unfolding of the 103-residue Fd. This predicted value is larger than the experimental one, indicating a more compact unfolded state (perhaps with local structure around the metal centers; see below) as compared with a random-coil polypeptide. For another iron–sulfur protein, the Ectothiorhodospira halophila high-potential iron–sulfur protein (similar size and structure as A. ambivalens Fd but from a mesophile), CP was found to be 4.5 kJ/(mole • K) (Iwagami et al. 1995), comparable to the value for A. ambivalens Fd.
A low CP corresponds to a shallow dependence of the GU on temperature. In addition, the stability for Fd at 20°C, pH 7 (Fig. 3) appears very high. Taken together, these results indicate that Fd achieves its increased thermostability through a combination of a high maximal GU (model one, see introduction) and a low CP (model three, see introduction). This combination of mechanisms to attain thermostability was also observed for the Thermus thermophilus RNase H protein (Hollien and Marqusee 1999), and several other investigations have supported the `flattened' third model (McCrary et al. 1996; Beadle et al. 1999; Hollien and Marqusee 1999). In contrast to the very high GU found at low temperatures, at the A. ambivalens optimal growth temperature around 80°C, the Fd stability at pH 7 is not more than 40 kJ/mole, a value similar to that for an average mesophilic protein at 20°C (Makhatadze and Privalov 1995).
Fd stability as a function of pH
The stability of A. ambivalens Fd is decreased at lower pHs (Fig. 4; Table 2), supporting the importance of electrostatic interactions for the overall stability. The abrupt decrease in stability at pH < 4 may indicate that interactions involving deprotonated Asp or Glu side chains are contributing to the high stability at neutral pH. At low pH, the free energy contribution associated with proton binding decreases the stability of the native conformation because of the favorable protonation of carboxylate groups (e.g., Asp or Glu) in the unfolded state (Luisi and Raleigh 2000). The most prominent pairwise ionic interactions in native Fd appear to be those between Asp 99–Arg 7 and Asp 41–Arg 10 (in the N-terminal region, the first pair directly linking the N and C termini) and also between Asp 64–Lys 73 and Asp 43–His 68 (see Fig. 6; Wittung-Stafshede et al. 2000). All of these residues are conserved among seven-iron ferredoxins from Sulfolobales (Gomes et al. 1998), perhaps indicating that such ion pairs, or the resulting overall electrostatic field, are common strategies to obtain thermostability in these proteins.
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Role of metal centers for Fd folding
Two iron–sulfur clusters are linked to the Fd polypeptide through eight cysteine sulfurs. Upon Fd unfolding at low pH, the cysteine sulfurs, involved in iron–sulfur cluster binding, may become protonated (and thus would contribute to the pH dependence of the stability). Using Ellman's assay we did not, however, detect any free sulfurs in the unfolded state of Fd, indicating that sulfur oxidation is promoted after Fd unfolding and metal cluster decomposition. Sulfur oxidation was shown to occur in oxidized azurin, a copper protein in which a cysteine sulfur is one metal ligand, upon GuHCl-induced protein unfolding and metal dissociation (Leckner et al. 1997), as well as upon iron release in desulforedoxin (in which iron coordinates four cysteine sulfurs) at low pH (Kennedy et al. 1998). The mechanism of cluster decomposition and subsequent cysteine oxidation in unfolded Fd may still occur through protonated cysteine intermediates. In fact, the strong pH dependence observed for the Fd stability indicates that at least some of the eight cysteine sulfurs become protonated after polypeptide unfolding and subsequent cluster decomposition. Ionic interactions on the protein's surface and the cysteine ligands of the iron–sulfur clusters may together explain the sharp pH dependence of Fd thermodynamic stability.
The residues involved in zinc coordination, three His and one Asp, are deprotonated in folded Fd but may become protonated upon low-pH unfolding, that is, if zinc dissociates. We revealed, however, that the zinc remains bound to the polypeptide upon ferredoxin unfolding (also at low pH). The same observation was made for zinc-substituted azurin: The zinc ion remained coordinated, presumably to some of the native-state ligands, also in the unfolded state (Leckner et al. 1997; Romero et al. 1998). According to the three-dimensional structural model (Wittung-Stafshede et al. 2000), the zinc in A. ambivalens Fd appears to hold the N-terminal region and the core fold together. In a recent study of the thermal behavior of truncated mutants of the Sulfolobus strain-7 ferredoxin (primary structure 95% identical to A. ambivalens Fd), also containing the zinc-binding site (Fujii et al. 1997), it was shown that the zinc was partly responsible for the thermal stabilization (Kojoh et al. 1999). It is possible that zinc coordination in the unfolded state (to some or all of the native-state ligands) decreases the entropy of this state; in this way the stability of the folded structure is increased. Residual structure, and/or a compact denatured state, is in accord with the low CP found upon Fd unfolding.
That the iron–sulfur clusters stabilize the native structure of Fd is clear from the observation that chemical and thermal unfolding processes (leading ultimately to cluster dissociation and degradation) are irreversible. Fd refolding attempts at pH 2.5 (but not at higher pHs) showed formation of a large fraction of secondary structure. However, no characteristic color appeared, indicating the presence of only apoprotein under these conditions. We found the "refolded" apoprotein to enhance ANS emission significantly (11x), as well as to have a noncooperative far-UV CD thermal profile (data not shown). Both results are in accord with a nonnative, molten-globule-like structure for the apoprotein at low pH. It was shown for the [2Fe-2S] spinach ferredoxin (Pagani et al. 1986) that (enzymatic) cluster assembly and insertion drive the polypeptide toward full recovery of the native form. Thus, without inserted iron–sulfur clusters, ferredoxin proteins appear incapable of adopting their native folds. This indicates that for ferredoxin formation in vivo, cluster insertion may precede polypeptide folding, or chaperone proteins may be required. There are several reports supporting that cofactors (e.g., heme, copper, flavin, iron–sulfur clusters) can remain coordinated to their corresponding polypeptides upon unfolding (Leckner et al. 1997; Wittung-Stafshede 1999; Apiyo et al. 2000; Moczygemba et al. 2000). It is thus possible that for several cofactor-binding proteins in vivo, coordination of cofactor(s) to the unfolded polypeptide occurs before folding takes place. The cofactor(s) may in this way aid in shifting the equilibrium toward a native-like ordered structure.
In a wide pH range, Fd unfolding first leads (within seconds) to a denatured state in which the polypeptide coordinate rearranged, possibly linear [3Fe-4S], iron clusters. On a longer time scale, the new clusters decompose, yielding unfolded apo-Fd. Rearrangements into linear three-iron clusters were shown for beef-heart aconitase at high pH (pH > 9.5) and at neutral pH in the presence of urea (Kennedy et al. 1984), as well as for another seven-iron Fd at pH 7 (in the presence of GuSCN; Fig. 5B). Unlike our results on the two thermostable ferredoxins, the linear [3Fe-4S] cluster in partly unfolded aconitase was stable for days at high pH and showed a half-time of 40 min in urea and pH 8 (Kennedy et al. 1984). Further studies may reveal if formation of this intermediate species is part of a general mechanism for cluster decomposition in iron–sulfur proteins.
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Protein preparation
A. ambivalens cells were grown in a 10-L fermentor as described previously (Teixeira et al. 1995). S. acidocaldarius cells were a kind gift from Prof. K.O. Stetter and H. Huber from Regensburg University, Germany. The A. ambivalens and S. acidocaldarius Fds were purified as detailed in Gomes et al. (1998). Protein purity was confirmed by a single band on 15% SDS–PAGE and a clean N-terminal sequence.
Denaturant-induced unfolding
Chemical denaturant GuHCl was used to promote protein unfolding (at 20°C) at all pHs except for pH 7; at the latter pH GuSCN was used. GuHCl/GuSCN titrations were performed at room temperature with 20 µM Fd in 5 mM phosphate (pH 7.0 and 5.2), Tris-HCl (pH 8.5), acetate (pH 4.0) or glycine (pH 2.5 and 10) buffer. There was no protein-concentration dependence for the unfolding transitions (in accord with apparent two-state transitions although irreversible processes; see text for discussion). Samples were incubated for 30 min before measurements (variation of the incubation time from 2 min to 24 h did not alter the observed transitions). Unfolding was monitored by far-UV CD (200–300 nm, 1-mm cell) on an OLIS spectropolarimeter, visible absorption (300–700 nm, 1-cm cell) on a Cary-50 spectrophotometer, and tryptophan emission (300–450 nm, excitation 280 nm, 1-cm cell) on a Varian Eclipse fluorometer.
Unfolding transitions were analyzed using a two-state model, fF + fU = 1, where fF and fU represent the fractions of total protein in the folded and denatured conformations, respectively. At each point in the reaction, the equilibrium constant, KU, and the free energy change, GU, are related as follows:
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GU can be expressed as a function of the denaturant concentration:
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In this equation, m describes the extent of hydrophobic surface exposure upon unfolding (Tanford 1970; Pace 1975) and GU(H2O) is the free energy of unfolding in aqueous solution. Direct fits (in KaleidaGraph) to the experimental unfolding curves were performed using the following expression, derived from equations 1, 2, and 3:
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Yobs, YU, and YF are the observed spectroscopic signal, denatured protein baseline, and folded protein baseline, respectively. From the fits, GU(H2O) was determined at each condition (Table 2). The transition midpoints were calculated as GU(H2O)/m or by direct inspection of the transitions. Reported uncertainties were obtained from the goodness of the fits.
Comparative GuHCl and GuSCN titrations were performed on sperm whale Met myoglobin; degree of unfolding was monitored by changes in heme absorption (409 nm) as reported previously (Moczygemba et al. 2000). The observed midpoints, [GuHCl]1/2 = 1.6 ± 0.1 M and [GuSCN]1/2 = 0.7 ± 0.1 M, were used to calculate a scaling factor of 2.3 for interconversion between GuHCl and GuSCN midpoint concentrations. This scaling factor is in excellent agreement with GuHCl and GuSCN data reported for barnase (Cota and Clarke 2000). The activity of GuSCN was shown to be linear up to at least 3.5 M; therefore, no correction of molar concentrations into activity units was performed (Pandya et al. 1999).
Thermally induced unfolding
Temperature-induced unfolding of Fd was monitored by far-UV CD (200–300 nm) in various pH/GuHCl conditions (see Table 1). CD spectra of 20 µM ferredoxin were recorded every 5°C from 20°C to 95°C, with 5 min of equilibrium at each temperature. Longer equilibration times (10 min) at each temperature did not change the melting profiles. In the end, the temperature was decreased to 20°C and a CD spectrum was recorded to check for refolding. The thermal reactions were monitored on the OLIS instrument connected to a digitally controlled water bath (Julabo). Analyses of thermal transitions according to a modified Van't Hoff equation (Makhatadze and Privalov 1995; Wittung-Stafshede et al. 1998), which accounted for the temperature dependence of H and S (first using a predicted Cp of 6 kJ/mole [Myers et al. 1995] followed by revision using a value of 3 kJ/mole), yielded H(Tm) at each Tm (Table 1).
Unfolding kinetics
Kinetic unfolding measurements (20°C) were made on an Applied Photophysics SX.18MV stopped-flow reaction analyzer; absorption mode (410 nm and 610 nm detection); 1 : 1 mixing of native Fd and 8 M GuHCl (buffered to pH 2.5); pathlength 2 mm. No amplitude changes occurred in the dead time (<2 msec) of the instrument. For each time range, a minimum of eight kinetic traces was averaged and fit to monophasic decay equations using a nonlinear least-squares algorithm supplied by Applied Photophysics.
Irreversibility of unfolding reactions
Fd unfolding is irreversible upon both thermal and chemical denaturant perturbations, as also reported earlier (Wittung-Stafshede et al. 2000). Experimental results and related literature supporting a thermodynamic treatment of the collected data despite the irreversibility are discussed throughout the text. The unfolded protein does not aggregate. Tests with Ellman's reagent (DTNB) show that at least some of the cysteine sulfurs, which coordinate to the iron–sulfur clusters in native Fd, also remain unavailable in the unfolded state (indicating sulfur oxidation). At low pH (but not at neutral and high pHs), 80% of the native amount of secondary structure was regained in refolding attempts (by dilution from high to low denaturant conditions, 20°C, monitored by far-UV CD). No characteristic color was observed for this species, indicating the presence of only apoprotein. This nonnative (but not fully unfolded) apo form of Fd was characterized in terms of ANS binding (fluorescent probe for exposed hydrophobic surfaces) and thermal melting (see Discussion).
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