Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific beta recombinase, and identification of a folding intermediate

Solution properties of beta recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) beta recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold beta recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N2-->2I-->2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to deltaG(tot) = 17.9 kcal/mol, with deltaG(N2-->2I) = 4.2 kcal/mol for the first transition and deltaG(I-->U) = 6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of beta recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate.     

Energetics of folding subtilisin BPN'.

Subtilisin is an unusual example of a monomeric protein with a substantial kinetic barrier to folding and unfolding. Here we document for the first time the in vitro folding of the mature form of subtilisin. Subtilisin was modified by site-directed mutagenesis to be proteolytically inactive, allowing the impediments to folding to be systematically examined. First, the thermodynamics and kinetics of calcium binding to the high-affinity calcium A-site have been measured by microcalorimetry and fluorescence spectroscopy. Binding is an enthalpically driven process with an association constant (Ka) equal to 7 x 10(6) M-1. Furthermore, the kinetic barrier to calcium removal from the A-site (23 kcal/mol) is substantially larger than the standard free energy of binding (9.3 kcal/mol). The kinetics of calcium dissociation from subtilisin (e.g., in excess EDTA) are accordingly very slow (t1/2 = 1.3 h at 25 degrees C). Second, to measure the kinetics of subtilisin folding independent of calcium binding, the high-affinity calcium binding site was deleted from the protein. At low ionic strength (I = 0.01) refolding of this mutant requires several days. The folding rate is accelerated almost 100-fold by a 10-fold increase in ionic strength, indicating that part of the free energy of activation may be electrostatic. At relatively high ionic strength (I = 0.5) refolding of the mutant subtilisin is complete in less than 1 h at 25 degrees C. We suggest that part of the electrostatic contribution to the activation free energy for folding subtilisin is related to the highly charged region of the protein comprising the weak ion binding site (site B).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dimeric procaspase-3 unfolds via a four-state equilibrium process.

We have examined the folding and assembly of a catalytically inactive mutant of procaspase-3, a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. The equilibrium unfolding data demonstrate a plateau between 3 and 5 M urea, consistent with an apparent three-state unfolding process. However, the midpoint of the second transition as well as the amplitude of the plateau are dependent on the protein concentration. Overall, the data are well described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric intermediate dissociates to a monomeric intermediate, which then unfolds. By fitting the four-state model to the experimental data, we have determined the free energy change for the first step of unfolding to be 8.3 +/- 1.3 kcal/mol. The free energy change for the dissociation of the dimeric folding intermediate to two monomeric intermediates is 10.5 +/- 1 kcal/mol. The third step in the unfolding mechanism represents the complete unfolding of the monomeric intermediate, with a free energy change of 7.0 +/- 0.5 kcal/mol. These results show two important points. First, dimerization of procaspase-3 occurs as a result of the association of two monomeric folding intermediates, demonstrating that procaspase-3 dimerization is a folding event. Second, the stability of the dimer contributes significantly to the conformational free energy of the protein (18.8 of 25.8 kcal/mol).     

Complex of human apolipoprotein C-1 with phospholipid: thermodynamic or kinetic stability?

Thermal unfolding of discoidal complexes of apolipoprotein (apo) C-1 with dimyristoyl phosphatidylcholine (DMPC) reveals a novel mechanism of lipoprotein stabilization that is based on kinetics rather than thermodynamics. Far-UV CD melting curves recorded at several heating/cooling rates from 0.047 to 1.34 K/min show hysteresis and scan rate dependence characteristic of slow nonequilibrium transitions. At slow heating rates, the apoC-1 unfolding in the complexes starts just above 25 degrees C and has an apparent melting temperature T(m) approximately 48 +/- 1.5 degrees C, close to T(m) = 51 +/- 1.5 degrees C of free protein. Thus, DMPC binding may not substantially increase the low apparent thermodynamic stability of apoC-1, DeltaG(25 degrees C) < 2 kcal/mol. The scan rate dependence of T(m) and Arrhenius analysis of the kinetic data suggest an activation enthalpy E(a) = 25 +/- 5 kcal/mol that provides the major contribution to the free energy barrier for the protein unfolding on the disk, DeltaG > or = 17 kcal/mol. Consequently, apoC-1/DMPC disks are kinetically but not thermodynamically stable. To explore the origins of this kinetic stability, we utilized dynode voltage measured in CD experiments that shows temperature-dependent contribution from UV light scattering of apoC-1/DMPC complexes (d approximately 20 nm). Correlation of CD and dynode voltage melting curves recorded at 222 nm indicates close coupling between protein unfolding and an increase in the complex size and/or lamellar structure, suggesting that the enthalpic barrier arises from transient disruption of lipid packing interactions upon disk-to-vesicle fusion. We hypothesize that a kinetic mechanism may provide a general strategy for lipoprotein stabilization that facilitates complex stability and compositional variability in the absence of high packing specificity.     

Role of the minor energetic determinants of chicken egg white lysozyme (HEWL) to the stability of the HEWL.antibody scFv-10 complex

Seven of the 13 non-glycine contact amino acids in the hen (chicken) egg white lysozyme (HEWL) epitope for antibody Fab-10 each contribute < or =0.3 kcal/mol to the change in free energy (DeltaDeltaG(D)) from wild type (WT) when replaced by alanine (nullspots), and three others each give (0.7 < DeltaDeltaG(D) < or = 1. 0) kcal/mol (warm spots) (Rajpal et al. Protein Sci 1998;7:1868-1874). The low DeltaDeltaG(D) values introduced by alanine mutations present an opportunity to explore accurately their cumulative effects, as the sum of the combined DeltaDeltaG(D) values is not so large as to destabilize the complex beyond the range of accurate measurement. Substitution of six of the seven null spot residues by alanine leads to a cumulative DeltaDeltaG(D) = 2.25 +/- 0.04 kcal/mol, whereas the sum of the six individual changes is only -0.36 +/- 0.32 kcal/mol. The triple warm spot mutation generates a DeltaDeltaG(D) = 5.11 +/- 0.06 kcal/mol versus DeltaDeltaG(D) = 2.52 +/- 0.22 kcal/mol for the sum of the three individuals. The non-additivity in the individual DeltaDeltaG(D) values for the alanine mutations may indicate that these residues provide a conformationally stabilizing effect on the hot spot residues, each of which exhibits DeltaDeltaG(D) > 4.0 kcal/mol on alanine substitution.     

Molecular Dynamics/Free Energy Perturbation Studies of the Thermostable V74I Mutant of Ribonuclease HI

Abstract

Recent site-directed mutagenesis and thermodynamic studies have shown that the V74I mutant of Escherichia coli ribonuclease HI (RNase HI) is more stable than the wild type protein [Ishikawa et al., Biochemistry 32, 6171 (1993)]. In order to clarify the stabilization mechanism of this mutant, we calculated the free energy change due to the mutation Val 74→Ile in both the native and denatured states by free energy perturbations based on molecular dynamics (MD) simulations. We carried out inclusive MD simulations for the protein in water; i.e., fully solvated, no artificial constraints applied, and all long-range Coulomb interactions included. We found that the free energy of the mutant increased slightly relative to the wild type, in the native state by 1.60 kcal/mol, and in the denatured state by 2.25 kcal/mol. The unfolding free energy increment of the mutant (0.66 ± 0.19 kcal/mol) was in good agreement with the experimental value (0.6 kcal/mol). The hysteresis error in the free energy calculations, i.e., forward and reverse perturbations, was only ±0.19 kcal/mol. These results show that the V74I mutant is stabilized relative to the wild type by the increased free energy of the denatured state and not by a decrease in the free energy of the native state as had been proposed earlier based on the mutant X-ray structure. It was found that the stabilization was caused by a loss of solvation energy in the mutant denatured state and not by improved packing interactions inside the native protein.     

The standard Gibbs free energy change of hydrolysis of alpha-D-ribose 1-phosphate

The standard Gibbs free energy change of hydrolysis of α-d-ribose 1-phosphate has been measured at pH 7.0, ionic strength 0.1 m, and 25 °C by combining the corresponding values of the two following reactions: adenosine + H₂O ág adenine + ribose (ΔG^0′ = ?2.3 ± 0.1 kcal/mol), catalyzed by adenosine nucleosidase, and ribose 1-phosphate + adenine ág adenosine + P_i (ΔG^0′ = ?3.1 ± 0.1 kcal/mol), catalyzed by adenosine phosphorylase. The standard Gibbs free energy changes were calculated for both reactions from the equilibrium constant. A value of -5.4 ± 0.15 kcal/mol, comparable to that of other hemiacetal phosphoric esters, was obtained for the hydrolysis of ribose 1-phosphate.     

Folding of a de novo designed native-like four-helix bundle protein

The folding of a model native-like dimeric four-helix bundle protein, (alpha(2))(2), was investigated using guanidine hydrochloride, hydrostatic pressure, and low temperature. Unfolding by guanidine hydrochloride followed by circular dichroism and intrinsic fluorescence spectroscopy revealed a highly cooperative transition between the native-like and unfolded states, with free energy of unfolding determined from CD data, DeltaG(unf) = 14.3 +/- 0.8 kcal/mol. However, CD and intrinsic fluorescence data were not superimposable, indicating the presence of an intermediate state during the folding transition. To stabilize the folding intermediate, we used hydrostatic pressure and low temperature. In both cases, dissociation of the dimeric native-like (alpha(2))(2) into folded monomers (alpha(2)) was observed. van't Hoff analysis of the low temperature experiments, assuming a two-state dimer 171-monomer transition, yielded a free energy of dissociation of (alpha(2))(2) of DeltaG(diss) = 11.4 +/- 0.4 kcal/mol, in good agreement with the free energy determined from pressure dissociation experiments (DeltaG(diss) = 10.5 +/- 0.1 kcal/mol). Binding of the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to the pressure- and cold-dissociated states of (alpha(2))(2) indicated the existence of molten-globule monomers. In conclusion, we demonstrate that the folding pathway of (alpha(2))(2) can be described by a three-state transition including a monomeric molten globule-like state.     

Thermodynamic and kinetic stability of a large multi-domain enzyme from the hyperthermophile Aeropyrum pernix

The multi-domain enzyme isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix was studied by denaturant-induced unfolding. At pH 7.5, changes in circular dichroism ellipticity and intrinsic fluorescence showed a complex unfolding transition, whereas at pH 3.0, an apparently two-state and highly reversible unfolding occurred. Analytical ultracentrifugation revealed the dissociation from dimer to monomer at pH 3.0. The thermodynamic and kinetic stability were studied at pH 3.0 to explore the role of inter-domain interactions independently of inter-subunit interplay on the wild type and R211M, a mutant where a seven-membered inter-domain ionic network has been disrupted. The unfolding and folding transitions occurred at slightly different denaturant concentrations even after prolonged equilibration time. The difference between the folding and the unfolding profiles was decreased in the mutant R211M. The apparent Gibbs free energy decreased approximately 2 kcal/mol and the unfolding rate increased 4.3-fold in the mutant protein, corresponding to a decrease in activation free energy of unfolding of 0.86 kcal/mol. These results suggest that the inter-domain ionic network might be responsible for additional stabilization through a significant kinetic barrier in the unfolding pathway that could also explain the larger difference observed between the folding and unfolding transitions of the wild type.     

Reciprocal cooperative effects of multiple ligand binding to pyruvate kinase

The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate.     

High- and low-temperature unfolding of human high-density apolipoprotein A-2.

Human plasma apolipoprotein A-2 (apoA-2) is the second major protein of the high-density lipoproteins that mediate the transport and metabolism of cholesterol. Using CD spectroscopy and differential scanning calorimetry, we demonstrate that the structure of lipid-free apoA-2 in neutral low-salt solutions is most stable at approximately 25 degrees C and unfolds reversibly both upon heating and cooling from 25 degrees C. High-temperature unfolding of apoA-2, monitored by far-UV CD, extends from 25-85 degrees C with midpoint Th = 56 +/- 2 degrees C and vant Hoff's enthalpy delta H(Th) = 17 +/- 2 kcal/mol that is substantially lower than the expected enthalpy of melting of the alpha-helical structure. This suggests low-cooperativity apoA-2 unfolding. The apparent free energy of apoA-2 stabilization inferred from the CD analysis of the thermal unfolding, delta G(app)(25 degrees) = 0.82 +/- 0.15 kcal/mol, agrees with the value determined from chemical denaturation. Enhanced low-temperature stability of apoA-2 observed upon increase in Na2HPO4 concentration from 0.3 mM to 50 mM or addition of 10% glycerol may be linked to reduced water activity. The close proximity of the heat and cold unfolding transitions, that is consistent with low delta G(app)(25 degrees), indicates that lipid-free apoA-2 has a substantial hydrophobic core but is only marginally stable under near-physiological solvent conditions. This suggests that in vivo apoA-2 transfer is unlikely to proceed via the lipid-free state. Low delta H(Th) and low apparent delta Cp approximately 0.52 kcal/mol.K inferred from the far-UV CD analysis of apoA-2 unfolding, and absence of tertiary packing interactions involving Tyr groups suggested by near-UV CD, are consistent with a molten globular-like state of lipid-free apoA-2.     

The thermotropic properties of human plasma fibronectin

Thermal denaturation profiles for human plasma fibronectin under a variety of conditions have been determined. Although a single melting curve for this protein, with a thermal transition midpoint of 58.4 +/- 1.0 degree C and a calorimetric enthalpy change (delta Hc) of 1040 +/- 100 kcal/mol, is obtained in dilute neutral salt solutions, it is estimated that a total of seven to eight independent two-state thermal transitions are present in this endotherm. These values are not significantly altered by the presence of Ca2+, up to levels of at least 20 mM. Upon variation of the pH, no distinct thermal transitions are noted at values below pH 5.0 and above pH 10.0. Between pH 7.0 and 10.0, virtually no alterations in the thermotropic properties of fibronectin are observed, indicating that the individual domains of this protein, which contribute to the thermogram, are preserved in this pH range. Upon alteration of the ionic strength of the buffer, from 0.05 to 0.4 M KCl, small changes are observed in the thermal transition profiles of fibronectin, indicative of conformational changes in the protein resulting in a larger number of cooperative units undergoing the temperature-induced unfolding reaction.     

Involvement of cysteine residues and domain interactions in the reversible unfolding of lipoxygenase-1

Urea-induced unfolding of lipoxygenase-1 (LOX1) at pH 7.0 was followed by enzyme activity, spectroscopic measurements, and limited proteolysis experiments. Complete unfolding of LOX1 in 9 M urea in the presence of thiol reducing or thiol modifying reagents was observed. The aggregation and oxidative reactions prevented the reversible unfolding of the molecule. The loss of enzyme activity was much earlier than the structural loss of the molecule during the course of unfolding, with the midpoint concentrations being 4.5 and 7.0 M for activity and spectroscopic measurements, respectively. The equilibrium unfolding transition could be adequately fitted to a three-state, two-step model (N left arrow over right arrow I left arrow over right arrow U) and the intermediate fraction was maximally populated at 6.3 M urea. The free energy change (DeltaG(H(2)O)) for the unfolding of native (N) to intermediate (I) was 14.2 +/- 0.28 kcal/mol and for the intermediate to the unfolded state (U) was 11.9 +/- 0.12 kcal/mol. The ANS binding measurements as a function of urea concentration indicated that the maximum binding of ANS was in 6.3 M urea due to the exposure of hydrophobic groups; this intermediate showed significant amount of tertiary structure and retained nearly 60% of secondary structure. The limited proteolysis measurements showed that the initiation of unfolding was from the C-terminal domain. Thus, the stable intermediate observed could be the C-terminal domain unfolded with exposed hydrophobic domain-domain interface. Limited proteolysis experiments during refolding process suggested that the intermediate refolded prior to completely unfolded LOX1. These results confirmed the role of cysteine residues and domain-domain interactions in the reversible unfolding of LOX1. This is the first report of the reversible unfolding of a very large monomeric, multi-domain protein, which also has a prosthetic group.     

Differential scanning calorimetry of thermal unfolding of the methionine repressor protein (MetJ) from Escherichia coli.

The thermal stability of the methionine repressor protein from Escherichia coli (MetJ) has been examined over a wide range of pH (pH 3.5-10) and ionic strength conditions using differential scanning calorimetry. Under reducing conditions, the transitions are fully reversible, and thermograms are characteristic of the cooperative unfolding of a globular protein with a molecular weight corresponding to the MetJ dimer, indicating that no dissociation of this dimeric protein occurs before unfolding of the polypeptide chains under most conditions. In the absence of reducing agent, repeated scans in the calorimeter show only partial reversibility, though the thermodynamic parameters derived from the first scans are comparable to those obtained under fully reversible conditions. The protein is maximally stable (Tm 58.5 degrees C) at about pH 6, close to the estimated isoelectric point, and stability is enhanced by increasing ionic strength in the range I = 0.01-0.4 M. The average calorimetric transition enthalpy (delta Hm) for the dimer is 505 +/- 28 kJ mol-1 under physiological conditions (pH 7, I = 0.125, Tm = 53.2 degrees C) and shows a small temperature dependence which is consistent with an apparent denaturational heat capacity change (delta Cp) of about +8.9 kJ K-1 mol-1. The effects of both pH and ionic strength on the transition temperature and free energy of MetJ unfolding are inconsistent with any single amino acid contribution and are more likely the result of more general electrostatic interactions, possibly including significant contributions from electrostatic repulsion between the like-charged monomers which can be modeled by a Debye-Hückel screened potential.     

Thermodynamics of the arginine kinase reaction.

The effect of temperature, pH, free [Mg(2+)], and ionic strength on the apparent equilibrium constant of arginine kinase (EC 2.7.3.3) was determined. At equilibrium, the apparent K' was defined as [see text] where each reactant represents the sum of all the ionic and metal complex species. The K' at pH 7.0, 1.0 mM free [Mg(2+)], and 0. 25 M ionic strength was 29.91 +/- 0.59, 33.44 +/- 0.46, 35.44 +/- 0. 71, 39.64 +/- 0.74, and 45.19 +/- 0.65 (n = 8) at 40, 33, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy (DeltaH degrees') is -8.19 kJ mol(-1), and the corresponding standard apparent entropy of the reaction (DeltaS degrees') is + 2. 2 J K(-1)mol(-1) in the direction of ATP formation at pH 7.0, free [Mg(2+)] =1.0 mM, ionic strength (I) =0.25 M at 25 degrees C. We further show that the magnitude of transformed Gibbs energy (DeltaG degrees ') of -8.89 kJ mol(-1) is mostly comprised of the enthalpy of the reaction, with 7.4% coming from the entropy TDeltaS degrees' term (+0.66 kJ mol(-1)). Our results are discussed in relation to the thermodynamic properties of its evolutionary successor, creatine kinase.     

Porcine beta-lactoglobulin chemical unfolding: identification of a non-native alpha-helical intermediate

The chemical unfolding behavior of porcine beta-lactoglobulin (PLG) has been followed at pH 2 and 6 in the presence of guanidinium hydrochloride. The PLG unfolding transition, monitored by tryptophan fluorescence, far and near UV circular dichroism and 1D-NMR, can be described by a three-state transition suggesting the presence of at least one intermediate state that appears to display an excess of non-native alpha-helical structures. The thermodynamic parameters, as determined through a global analysis fitting procedure, give estimates of the free energy differences of the transitions connecting the native, the intermediate and the unfolded state: DeltaG(NI) (0) = 2.8 +/- 0.7 kcal mol(-1) (pH 2) and 4.2 +/- 0.5 kcal mol(-1) (pH 6) and DeltaG(NU) (0) = 7.2 +/- 0.6 kcal mol(-1) (pH 2) and 6.9 +/- 0.6 kcal mol(-1) (pH 6). CD unfolding data of the bovine species (BLG) have been collected here under the same experimental conditions of PLG to allow a careful comparison of the two beta-lactoglobulins. Intermediates with different characteristics have been identified for BLG and PLG, and their nature has been discussed on a structural analysis basis. The thermodynamic data reported here for PLG and BLG and the comparative analysis with data reported for equine beta lactoglobulin, show that homologous beta-barrel proteins, belonging to the same family and displaying high sequence identity (52-64%) populate unfolding intermediates to different extents, even though a common tendency to the formation of non-native alpha-helical intermediates, can be envisaged. The present results provide a prerequisite foundation of knowledge for the design and interpretation of future folding kinetic studies.     

A new method for determining the heat capacity change for protein folding

In order to use results from calorimetry or thermal unfolding curves to estimate the free energy change for protein unfolding at 25 degrees C, it is necessary to know the change in heat capacity for unfolding, delta Cp. We describe a new method for measuring delta Cp which is based on results from urea and thermal unfolding curves but does not require a calorimeter. We find that delta Cp = 1650 +/- 200 cal/(deg.mol) for the unfolding of ribonuclease T1 and that delta Cp = 2200 +/- 300 cal/(deg.mol) for the unfolding of ribonuclease A.     

The unfolding pathway for Apo Escherichia coli aspartate aminotransferase is dependent on the choice of denaturant

The guanidine hydrochloride (GdnHCl) mediated denaturation pathway for the apo form of homodimeric Escherichia coli aspartate aminotransferase (eAATase) (molecular mass = 43.5 kDa/monomer) includes a partially folded monomeric intermediate, M* [Herold, M., and Kirschner, K. (1990) Biochemistry 29, 1907-1913; Birolo, L., Dal Piaz, F., Pucci, P., and Marino, G. (2002) J. Biol. Chem. 277, 17428-17437]. The present investigation of the urea-mediated denaturation of eAATase finds no evidence for an M* species but uncovers a partially denatured dimeric form, D*, that is unpopulated in GdnHCl. Thus, the unfolding process is a function of the employed denaturant. D* retains less than 50% of the native secondary structure (circular dichroism), conserves significant quaternary and tertiary interactions, and unfolds cooperatively (mD*<==>U = 3.4 +/- 0.3 kcal mol-1 M-1). Therefore, the following equilibria obtain in the denaturation of apo-eAATase: D <==> 2M 2M* <==> 2U in GdnHCl and D <==> D* <==> 2U in urea (D = native dimer, M = folded monomer, and U = unfolded state). The free energy of unfolding of apo-eAATase (D <==> 2U) is 36 +/- 3 kcal mol-1, while that for the D* 2U transition is 24 +/- 2 kcal mol-1, both at 1 M standard state and pH 7.5.     

Conformational stability and mechanism of folding of ribonuclease T1

Urea and thermal unfolding curves for ribonuclease T1 (RNase T1) were determined by measuring several different physical properties. In all cases, steep, single-step unfolding curves were observed. When these results were analyzed by assuming a two-state folding mechanism, the plots of fraction unfolded protein versus denaturant were coincident. The dependence of the free energy of unfolding, delta G (in kcal/mol), on urea concentration is given by delta G = 5.6 - 1.21 (urea). The parameters characterizing the thermodynamics of unfolding are: midpoint of the thermal unfolding curve, Tm = 48.1 degrees C, enthalpy change at Tm, delta Hm = 97 kcal/mol, and heat capacity change, delta Cp = 1650 cal/mol deg. A single kinetic phase was observed for both the folding and unfolding of RNase T1 in the transition and post-transition regions. However, two slow kinetic phases were observed during folding in the pre-transition region. These two slow phases account for about 90% of the observed amplitude, indicating that a faster kinetic phase is also present. The slow phases probably result from cis-trans isomerization at the 2 proline residues that have a cis configuration in folded RNase T1. These results suggest that RNase T1 folds by a highly cooperative mechanism with no structural intermediates once the proline residues have assumed their correct isomeric configuration. At 25 degrees C, the folded conformation is more stable than the unfolded conformations by 5.6 kcal/mol at pH 7 and by 8.9 kcal/mol at pH 5, which is the pH of maximum stability. At pH 7, the thermodynamic data indicate that the maximum conformational stability of 8.3 kcal/mol will occur at -6 degrees C.