Stability and folding of the ferredoxin from the hyperthermophilic archaeon Acidianus ambivalens

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric protein containing two iron-sulfur centres, one [3Fe-4S](1+/0) and one [4Fe-4S](2+/1+). It is an intrinsically hyperstable protein, being expressed at the organism's extreme optimal growth temperature: 80 degrees C. Using spectroscopic methods we have investigated the unfolding reaction of the Acidianus ambivalens ferredoxin. No unfolding of the oxidised ferredoxin was observed at pH 7.0, even in the presence of 8 M GuHCl. Upon increasing the pH to 10.0, the unfolding transition showed a midpoint at 6.3 M GuHCl and an unfolding-free energy of 70 kJ mol(-1) in buffer (pH 10) was estimated. Kinetic-unfolding experiments showed that the polypeptide unfolding correlated with rearrangement of the iron-sulfur centres to new ones which had strong absorption maxima at 520 and 610 nm. These new, possibly linear three-iron, clusters were coordinated to the unfolded protein but degraded slowly. From thermal experiments in the presence of GuHCl we estimated the melting temperature for the Acidianus ambivalens ferredoxin in buffer (at pH 7) to be 122 degrees C. Possible structural properties that contribute to the large thermal stability of the Acidianus ambivalens ferredoxin are discussed using a three-dimensional protein model.     

High thermal and chemical stability of Thermus thermophilus seven-iron ferredoxin. Linear clusters form at high pH on polypeptide unfolding.

To probe the stability of the seven-iron ferredoxin from Thermus thermophilus (FdTt), we investigated its chemical and thermal denaturation processes in solution. As predicted from the crystal structure, FdTt is extremely resistant to perturbation. The guanidine hydrochloride-induced unfolding transition shows a midpoint at 6.5 m (pH 7, 20 degrees C), and the thermal midpoint is above boiling, at 114 degrees C. The stability of FdTt is much lower at acidic pH, suggesting that electrostatic interactions are important for the high stability at higher pH. On FdTt unfolding at alkaline pH, new absorption bands at 520 nm and 610 nm appear transiently, resulting from rearrangement of the cubic clusters into linear three-iron species. A range of iron-sulfur proteins has been found to accommodate these novel clusters in vitro, although no biological function has yet been assigned.     

A spectroscopic study of the temperature induced modifications on ferredoxin folding and iron-sulfur moieties

Thermal perturbation of the dicluster ferredoxin from Acidianus ambivalens was investigated employing a toolbox of spectroscopic methods. FTIR and visible CD were used for assessing changes of the secondary structure and coarse alterations of the [3Fe4S] and [4Fe4S] cluster moieties, respectively. Fine details of the disassembly of the metal centers were revealed by paramagnetic NMR and resonance Raman spectroscopy. Overall, thermally induced unfolding of AaFd is initiated with the loss of -helical content at relatively low temperatures (T(app)(m) approximately 44 degrees C), followed by the disruption of both iron-sulfur clusters (T(app)(m) approximately 53-60 degrees C). The degradation of the metal centers triggers major structural changes on the protein matrix, including the loss of tertiary contacts (T(app)(m) approximately 58 degrees C) and a change, rather than a significant net loss, of secondary structure (T(app)(m) approximately 60 degrees C). This latter process triggers a secondary structure reorganization that is consistent with the formation of a molten globule state. The combined spectroscopic approach here reported illustrates how changes in the metalloprotein organization are intertwined with disassembly of the iron-sulfur centers, denoting the conformational interplay of the protein backbone with cofactors.     

The effect of carbohydrate on the structure and stability of erythropoietin.

Erythropoietin is a glycoprotein hormone that stimulates the maturation of late erythroid progenitor cells. It has three N-linked and one O-linked carbohydrates which play an important role in the biosynthesis and biological activities of the protein. To determine the role the carbohydrate might have in maintaining the conformational stability of the protein, the protein expressed in mammalian cells (fully glycosylated), the asialo mammalian-expressed protein, and the protein expressed in Escherichia coli (no carbohydrate) were compared for their stability to guanidine HCl, pH, and temperature. Circular dichroism was used to follow protein unfolding. Both the intact and asialo mammalian-expressed proteins unfolded with a cooperative transition in guanidine HCl, with a midpoint at 1.75 M guanidine HCl. The E. coli-expressed material unfolded with a midpoint of 1.2 M guanidine HCl, and a delta G of unfolding which was 1.4 kcal/mol less than that of the two glycosylated molecules. The E. coli-derived protein was also significantly less stable to pH-induced conformational changes, showing a cooperative transition in 35% glycerol with a midpoint at pH 4.4, while both the intact and asialo mammalian-expressed molecules had a transition midpoint of pH 3.75 in the absence of glycerol, and approximately pH 3 in the presence of 35% glycerol. The E. coli-expressed molecule unfolded and precipitated upon heating to 44 degrees C, while the asialo and intact mammalian-expressed proteins remained soluble, with a Tm of 56 degrees C. From these experiments, the carbohydrate appears to play a critical role in stabilizing the erythropoietin molecule to denaturing conditions, and this increased stability does not depend on the presence of sialic acid.     

Role of amino-terminal residues in the folding of the constant fragment of the immunoglobulin light chain

Three constant fragments with different amino terminals, CL(105-214), CL(109-214), and CL(113-214), were obtained by limited proteolysis with trypsin or papain of a type lambda immunoglobulin light chain. The conformations of the three CL fragments were indistinguishable on the basis of circular dichroism and tryptophyl fluorescence spectra. The stability to heat and guanidine hydrochloride of CL(105-214) was almost the same as that of CL(109-214), but the stability of CL(113-214) was slightly lower than that of CL(105-214) or CL(109-214). The midpoint of the thermal unfolding transition at pH 7.5 was at 60.0 degrees C for CL(105-214), 60.4 degrees C for CL(109-214), and 57.5 degrees C for CL(113-214). The midpoint of the unfolding transition by guanidine hydrochloride at pH 7.5 and 25 degrees C was 1.2 M for CL(105-214) and CL(109-214) and at 1.0 M for CL(113-214). The kinetics of unfolding and refolding by guanidine hydrochloride of these CL fragments were analyzed on the basis of the three-species mechanism, U1 in equilibrium with U2 in equilibrium with N, where U1 and U2 are the slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. It was found that only the microscopic unfolding rate constant for CL(113-214) is 2-3 times greater than that for CL(105-214) or CL(109-214) and that the other microscopic rate constants for the three CL fragments are all the same. These findings indicated that the amino-terminal residues, Gly-109-Lys-112, or a part of them, stabilize the CL(113-214) fragment by decreasing only the unfolding rate, that the transition state of the folding of the CL fragment is independent of the presence or absence of this peptide, and that, at the last step of folding, the peptide is incorporated into the globular domain, thus stabilizing it.     

Unfolding thermodynamics of the tetrameric chaperone, SecB

SecB is a cytosolic tetrameric chaperone in Escherichia coli, which maintains polypeptides, destined for export in a translocation competent state. The thermodynamics of unfolding of SecB was studied as a function of protein concentration, by using high sensitivity-differential scanning calorimetry and spectroscopic methods. The thermal unfolding of tetrameric SecB is reversible and can be well described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers. Increasing the pH decreases the stability of the tetramer significantly, the T(m) changing from 341.3 K at pH 6.5 to 332.6 K at pH 9.5. The value of DeltaC(p) obtained from measurements of DeltaH(m) as a function of T(m) was 10.7 +/- 0.7 kcal mol(-1) K(-1). The value of DeltaC(p) is among the highest measured for a multimeric protein. At 298 K, pH 7.4, the DeltaG degrees (u) for the SecB tetramer is 27.9 +/- 2 kcal mol(-1). Denaturant-mediated unfolding of SecB was found to be irreversible. The reactivity of the four solvent-exposed free thiols in tetrameric SecB is salt dependent. The kinetics of reactivity suggests that these four cysteines are in close proximity to each other and that these residues on each monomer are in chemically identical environments. The thermodynamic data suggest that SecB is a stable, well-folded, and tightly packed tetramer and that substrate binding occurs at a surface site rather than at an interior cavity.     

Conformation and stability of elastase from Atlantic cod, Gadus morhua

Metal binding and conformational stability characteristics of psychrophilic elastase (ACE) from Atlantic cod (Gadus morhua) has been investigated. Chelation to Ca(2+) was found to be important for maintaining the biologically active conformation and for the thermal stability of the enzyme. However, presence of metal ions such as Zn(2+), Fe(3+) and Cu(2+) was found to inhibit its hydrolytic activity and so did the chelating agent EDTA. Both pH and guanidinium chloride induced denaturation of the enzyme was followed by monitoring the changes in the tryptophan fluorescence. ACE exhibited a simple two-state unfolding pattern in both acidic and basic conditions with the midpoint of transition at pH values 4.08 and 10.29, respectively. Guanidinium chloride and heat induced denaturation of the enzyme was investigated at two pH values, 5.50 and 8.00, wherein the enzyme possesses similar tertiary structure but differ in its hydrolytic activity. Guanidinium chloride induced denaturation indicated that the enzyme unfolds with a C(m) of 1.53 M at pH 8.0 and a DeltaG(H2O) of 6.91 kJ mol(-1) (28.65 J mol(-1) residue(-1)) which is the lowest reported for psychrophilic enzymes investigated till-date. However, at pH 5.50, DeltaG(H2O) value is slightly lowered by 0.65 kJ mol(-1) consistent with the observed increase in the apparent quenching constant obtained with acrylamide. On the other hand, increase in T(m) by 38.45 degrees C was observed for the enzyme at acid pH (5.50) in comparison to the heat induced unfolding at pH 8.0. The increase in the apparent T(m) has been attributed to the possible weak intermolecular association of the enzyme molecules at moderately high temperatures that is favoured by the increase in the accessible surface area / dynamics under acidic conditions. The stability characteristics of ACE have been compared with the available data for mesophilic porcine pancreatic elastase and possible mechanism for the low temperature adaptation of ACE has been proposed.     

On the relative contribution of ionic interactions over iron-sulfur clusters to ferredoxin stability

Metal centres play an important structural role in maintaining the native conformation of a protein. Here we use biophysical methods to investigate what is the relative contribution of iron-sulfur clusters in respect to ionic interactions in a thermophilic di-cluster ferredoxin model. Changes in protonation affect both the stability and the conformational dynamics of the protein fold. In the pH 5.5-8 interval, the protein has a high melting temperature (T(m) approximately 120 degrees C), which decreases towards pH extremes. Acidification triggers events in two steps: down to the isoelectric point (pH 3.5) the Fe-S clusters remain unchanged, the secondary structure content increases and the single Trp becomes more solvent shielded, denoting a more compact fold. Further acidification down to pH 2 sets off exposure of the hydrophobic core and Fe-S cluster disintegration, yielding a molten globule state. The relative stabilising contribution of the clusters becomes evident when stabilising ionic interactions are switched off as a result of poising the protein at pH 3.5, at an overall null charge: under these conditions, the Fe-S clusters disassemble at T(m)=72 degrees C, whereas the protein unfolds at T(m)=52 degrees C. Overall, this ferredoxin denotes a considerable structural plasticity around its native conformation, a property which appears to depend more on the integrity of its metal clusters rather than on the status of its stabilising electrostatic interactions. The latter however play a relevant role in determining the protein thermal stability.     

Antioxidant activity of sugar-lysine Maillard reaction products in cell free and cell culture systems

The presence of a linear [3Fe-4S] cluster in a protein was first observed in beef-heart aconitase. Here, we report the formation of linear [3Fe-4S] clusters upon guanidine hydrochloride (GuHCl)-induced unfolding of Aquifex aeolicus [2Fe-2S] ferredoxins (Fd) (AaeFd1, AaeFd4, and AaeFd5) at alkaline conditions (pH 10, 20 degrees C). We find the mechanism of linear [3Fe-4S] cluster formation to depend critically on the speed of polypeptide unfolding. In similarity to seven-iron Fds, polypeptide unfolding determines the rate by which linear [3Fe-4S] clusters form in AaeFd4 and AaeFd5. In contrast, in a disulfide-lacking variant of AaeFd1, which unfolds faster than AaeFd4 and AaeFd5, the polypeptides unfold first and the majority of clusters decompose. Next, unfolded polypeptides retaining intact clusters scavenge iron and sulfur to form linear [3Fe-4S] clusters in a bimolecular reaction. Wild-type AaeFd1 unfolds slower than the speed of linear-cluster decomposition, and the linear species is never populated. Linear [3Fe-4S] clusters may be intermediates during folding of iron-sulfur proteins.     

Kinetic traps in the folding/unfolding of procaspase-1 CARD domain

We have examined the folding and unfolding of the caspase recruitment domain of procaspase-1 (CP1-CARD), a member of the alpha-helical Greek key protein family. The equilibrium folding/unfolding of CP1-CARD is described by a two-state mechanism, and the results show CP1-CARD is marginally stable with a DeltaG(H2O) of 1.1 +/- 0.2 kcal/mole and an m-value of 0.65 +/- 0.06 kcal/mole/M (10 mM Tris-HCl at pH 8.0, 1 mM DTT, 25 degrees C). Consistent with the equilibrium folding data, CP1-CARD is a monomer in solution when examined by size exclusion chromatography. Single-mixing stopped-flow refolding and unfolding studies show that CP1-CARD folds and unfolds rapidly, with no detectable slow phases, and the reactions appear to reach equilibrium within 10 msec. However, double jump kinetic experiments demonstrate the presence of an unfolded-like intermediate during unfolding. The intermediate converts to the fully unfolded conformation with a half-time of 10 sec. Interrupted refolding studies demonstrate the presence of one or more nativelike intermediates during refolding, which convert to the native conformation with a half-time of about 60 sec. Overall, the data show that both unfolding and refolding processes are slow, and the pathways contain kinetically trapped species.     

Ferredoxins from the archaeon Acidianus ambivalens: overexpression and characterization of the non-zinc-containing ferredoxin FdB.

Two ferredoxin genes, fdA and fdB, from the extremely thermoacidophilic crenarchaeon Acidianus ambivalens have been sequenced; the sequences share 86% similarity. Whereas the deduced protein sequence of the ferredoxin FdA clearly contains a zinc-binding motif, the corresponding sequence of the FdB is devoid of this motif. Thus far, only the zinc-containing ferredoxin, FdA, from A. ambivalens has been chemically and functionally characterized from its native source. Using RT-PCR and Northern blot analysis, we show that both ferredoxins are expressed by A. ambivalens under either anaerobic or aerobic growth conditions. The zinc-free ferredoxin, FdB, was overexpressed in E. coli and purified to homogeneity. Using EPR spectroscopy, we could demonstrate that FdB contains one [3Fe-4S](1+/0) and one [4Fe-4S](2+/1+) cluster. The reduction potential of the [3Fe-4S](1+/0) cluster was determined as -235+/-10 mV, at pH 6.5, by EPR-monitored redox titration. The high melting temperature of 108+/-2 degrees C of FdB determined by CD spectroscopy reveals that it is not the binding of the Zn2+ that induces the extreme thermostability of these ferredoxins.     

Unfolding thermodynamics of Trp-cage, a 20 residue miniprotein, studied by differential scanning calorimetry and circular dichroism spectroscopy

Small proteins provide convenient models for computational studies of protein folding and stability, which are usually compared with experimental data. Until recently, the unfolding of Trp-cage was considered to be a two-state process. However, no direct experimental evidence for this has been presented, and in some cases, the contrary has been suggested. To elucidate a detailed unfolding mechanism, we studied the thermodynamics of unfolding of Trp-cage by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. The observation that at low temperatures only approximately 90-95% of Trp-cage exists in the native conformation presented an analytical challenge. Nevertheless, it was found that the DSC and CD data can be fitted simultaneously to the same set of thermodynamic parameters. The major uncertainty in such a global fit is the heat capacity change upon unfolding, DeltaCp. This can be circumvented by obtaining DeltaCp directly from the difference between heat capacity functions of the native and unfolded states. Using such an analysis it is shown that Trp-cage unfolding can be represented by a two-state model with the following thermodynamic parameters: Tm = 43.9 +/- 0.8 degrees C, DeltaH(Tm) = 56 +/- 2 kJ/mol, DeltaCp = 0.3 +/- 0.1 kJ/(mol.K). Using these thermodynamic parameters it is estimated that Trp-cage is marginally stable at 25 degrees C, DeltaG(25 degrees C) = 3.2 +/- 0.2 kJ/mol, which is only 30% more than the thermal fluctuation energy at this temperature.     

Pressure equilibrium and jump study on unfolding of 23-kDa protein from spinach photosystem II

Pressure-induced unfolding of 23-kDa protein from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20 degrees C and pH 5.5, 23-kDa protein shows a reversible two-state unfolding transition under pressure with a midpoint near 160 MPa, which is much lower than most natural proteins studied to date. The free energy (DeltaG(u)) and volume change (DeltaV(u)) for the unfolding are 5.9 kcal/mol and -160 ml/mol, respectively. It was found that NaCl and sucrose significantly stabilize the protein from unfolding and the stabilization is associated not only with an increase in DeltaG(u) but also with a decrease in DeltaV(u). The pressure-jump studies of 23-kDa protein reveal a negative activation volume for unfolding (-66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that, in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of 23-kDa protein are closer to each other and they are larger than that of the native state. The diverse pressure-refolding pathways of 23-kDa protein in some conditions were revealed in pressure-jump kinetics.     

Analysis of folding and unfolding reactions of cytochrome b5

The guanidine hydrochloride- (GuHCl-) induced unfolding and refolding of a recombinant domain of bovine microsomal cytochrome b(5) containing the first 104 amino acid residues has been characterized by both transient and equilibrium spectrophotometric methods. The soluble domain is reversibly unfolded and the equilibrium reaction may be monitored by changes in absorbance and fluorescence that accompany denaturation of the native protein. Both probes reveal a single cooperative transition with a midpoint at 3 M GuHCl and lead to a value for the protein stability (DeltaG(uw)) of 26.5 kJ mol(-1). This stability is much higher than that reported for the corresponding form of the apoprotein (approximately 7 kJ mol(-1)). Transient changes in fluorescence and absorbance during protein unfolding exhibit biphasic profiles. A fast phase occupying approximately 30% of the total amplitude is observed at high denaturant concentrations and becomes the dominant process within the transition region. The rates associated with each process show a linear dependency on GuHCl concentration, and at zero denaturant concentration the unfolding rates (k(uw)) are 4.5 x 10(-5) s(-1) and 5.2 x 10(-6) s(-1) at 25 degrees C. The pattern of unfolding is not correlated with covalent heterogeneity, since a wide range of variants and site-directed mutants exhibit identical profiles, nor is the unfolding correlated with cis-trans Pro isomerization in the native state. In comparison with the apo form of cytochrome b(5), the kinetics of refolding and unfolding are more complex and exhibit very different transition states. The data support a model for unfolding in which heme-protein interactions give rise to two discernible rates of unfolding. From an analysis of the activation parameters associated with each process it is established that two structurally similar transition states differing by less than 5 kJ mol(-1) exist in the unfolding reaction. Protein refolding exhibits monophasic kinetics but with distinct curvature apparent in plots of ln k(obs) versus denaturant concentration. The data are interpreted in terms of alternative routes for protein folding in which a "fast track" leads to the rapid ordering of structure around Trp26 for refolding while a slower route requires additional reorganization around the hydrophobic core.     

Exceptional stability of a [2Fe-2S] ferredoxin from hyperthermophilic bacterium Aquifex aeolicus

Aquifex aeolicus is the only hyperthermophile that is known to contain a plant- and mammalian-type [2Fe-2S] ferredoxin (Aae Fd1). This unique protein contains two cysteines, in addition to the four that act as ligands of the [2Fe-2S] cluster, which form a disulfide bridge. We have investigated the stability of Aae Fd1 with (wild-type) and without (C87A variant) the disulfide bond, with respect to pH, thermal and chemical perturbation, and compared the results to those for the mesophilic [2Fe-2S] ferredoxin from spinach. Unfolding reactions of all three proteins are irreversible due to cluster decomposition in the unfolded state. Wild-type and C87A Aae Fd1 proteins are extremely stable: unfolding at 20 degrees C requires high concentrations of the chemical denaturant and long incubation times. Moreover, their thermal-unfolding midpoints are 40-50 degrees higher than that for spinach ferredoxin (pH 7). The stability of the Aae Fd1 protein is significantly lower at pH 2.5 than pH 7 and 10, suggesting that ionic interactions play a role in structural integrity. Interestingly, the iron-sulfur cluster in C87A Aae Fd1 rearranges into a transient species with absorption bands at 520 and 610 nm, presumably a linear three-iron cluster, in the high-pH unfolded state.     

Thermal and conformational stability of seed coat soybean peroxidase

Soybean peroxidase (SBP) obtained from the soybean seed coats belongs to class III of the plant peroxidase superfamily. Detailed circular dichroism and steady state fluorescence studies have been carried out to monitor thermal as well as denaturant-induced unfolding of SBP and apo-SBP. Melting of secondary and tertiary structures of SBP occurs with characteristic transition midpoints, T(m), of 86 and 83.5 degrees C, respectively, at neutral pH. Removal of heme resulted in greatly decreased thermal stability of the protein (T(m) = 38 degrees C). The deltaG degrees (H2O) determined from guanidine hydrochloride-induced denaturation at 25 degrees C and at neutral pH is 43.3 kJ mol(-1) for SBP and 9.0 kJ mol(-1) for apo-SBP. Comparison with the reported unfolding data of the homologous enzyme, horseradish peroxidase (HRP-C), showed that SBP exhibits significantly high thermal and conformational stability. We show that this enhanced structural stability of SBP relative to HRP-C arises due to the unique nature of their heme binding. A stronger heme-apoprotein affinity probably due to the interaction between Met37 and the C8 heme vinyl substituent contributes to the unusually high structural stability of SBP.     

A hyperthermophilic plant-type [2Fe-2S] ferredoxin from Aquifex aeolicus is stabilized by a disulfide bond

A [2Fe-2S] ferredoxin (Fd1) from the hyperthermophilic bacterium Aquifex aeolicus has been obtained by heterologous expression of the encoding gene in Escherichia coli. Sequence comparisons show that this protein belongs to the extended family of plant- and mammalian-type [2Fe-2S] ferredoxins but also indicate that it is not closely similar to either the plant-type or mammalian-type subfamilies. Instead, it appears to bear some similarity to novel members of this family, in particular the Isc-type ferredoxins involved in the assembly of iron-sulfur clusters in vivo. The two redox levels of the [2Fe-2S](2+/+) metal site of A. aeolicus ferredoxin have been studied by UV-visible, resonance Raman, EPR, variable temperature magnetic circular dichroism, and M?ssbauer spectroscopies. A full-spin Hamiltonian analysis is given for the M?ssbauer spectra. In aggregate, the spectroscopic data reveal differences with both the plant-type and mammalian-type ferredoxins, in keeping with the sequence comparisons. The midpoint potential of the [2Fe-2S](2+/+) couple, at -375 mV versus the normal hydrogen electrode, is more negative than those of mammalian-type ferredoxins and at the upper end of the range covered by plant-type ferredoxins. A. aeolicus ferredoxin contains two cysteines in addition to the four that are committed as ligands of the [2Fe-2S] cluster. These two residues have been shown by chemical modification and site-directed mutagenesis to form a disulfide bridge in the native protein. While that cystine unit plays a significant role in the exceptional thermostability of A. aeolicus ferredoxin (T(m) = 121 degrees C at pH 7 versus T(m) = 113 degrees C in a molecular variant where the disulfide bridge has been removed), it does not bear on the properties of the [2Fe-2S](2+/+) chromophore. This observation is consistent with the large distance (ca. 20 A) that is predicted to separate the iron-sulfur chromophore from the disulfide bridge.     

Equilibrium unfolding of dimeric and engineered monomeric forms of lambda Cro (F58W) repressor and the effect of added salts: evidence for the formation of folded monomer induced by sodium perchlorate

The equilibrium unfolding transitions of Cro repressor variants, dimeric variant Cro F58W and monomer Cro K56[DGEVK]F58W, have been studied by urea and guanidine hydrochloride to probe the folding mechanism. The unfolding transitions of a dimeric variant are well described by a two state process involving native dimer and unfolded monomer with a free energy of unfolding, DeltaG(0,un)(0), of approximately 10-11 kcal/mol. The midpoint of transition curves is dependent on total protein concentration and DeltaG(0,un)(0) is independent of protein concentration, as expected for this model. Unfolding of Cro monomer is well described by the standard two state model. The stability of both forms of protein increases in the presence of salt but decreases with the decrease in pH. Because of the suggested importance of a N2<-->2F dimerization process in DNA binding, we have also studied the effect of sodium perchlorate, containing the chaotropic perchlorate anion, on the conformational transition of Cro dimer by CD, fluorescence and NMR (in addition to urea and guanidine hydrochloride) in an attempt both to characterize the thermodynamics of the process and to identify conditions that lead to an increase in the population of the folded monomers. Data suggest that sodium perchlorate stabilizes the protein at low concentration (<1.5 M) and destabilizes the protein at higher perchlorate concentration with the formation of a "significantly folded" monomer. The tryptophan residue in the "significantly folded" monomer induced by perchlorate is more exposed to the solvent than in native dimer.