Parallel stranded DNA with AT base pairing

The concentration and temperature dependences of the UV and CD spectra of the oligonucleotide 3'-d(ApTpApTpApTpApTpApTp)-O(CH2)6O-5'-d(pApTpApTpApTpApT pApT) (eicosamer) in aqueous solution at pH 7 in the presence of 0.5 M NaCl were studied. At less than 10(-6) M, the eicosamer was shown to form in solution a hairpin with parallel orientation of chains (parallel hairpin). From thermal denaturation profiles [A260(T)] the thermodynamic parameters, delta H degrees, delta S degrees and Tm for parallel hairpin formation were calculated to be -90 +/- 8 kJ/mol. -300 +/- 20 J.mol-1.K-1 and 40.5 degrees C, respectively. The CD spectra of the parallel double helix differed from those of B-form DNA and had characteristic features: decreasing magnitude of the positive maximum at 265 nm and a negative peak at 285 nm.     

Parallel double helical DNA. 1. Evidence for the existence of a spiral A-T-paired base

The dependence of UV and CD spectra of oligonucleotide 3'-d(ApTpApTpApTpApTpApTp)-O(CH2)6O-5'-(pApTpApTpApTpApTp ApT) (eicosamer) in aqueous solution at pH 7 in the presence of 0.5 M NaCl on temperature and concentration was studied. It was shown that the eicosamer in concentrations below 5.10(-4) M forms a parallel stranded hairpin. From the thermal denaturation profile the thermodynamic parameters of parallel hairpin formation were determined. The values of delta H0, delta S0 and Tm were -90 +/- 8 kJ/mol, -300 +/- 20 J.mol-1.K-1 and 40.5 degrees C, respectively. The CD spectra of the parallel helix differ from those of B-form DNA by reduction of extreme magnitude at approximately 265 nm and appearance of a negative effect at approximately 285 nm.     

Fluorescence and FTIR study of the pressure-induced denaturation of bovine pancreas trypsin.

The pressure denaturation of trypsin from bovine pancreas was investigated by fluorescence spectroscopy in the pressure range 0. 1-700 MPa and by FTIR spectroscopy up to 1000 MPa. The tryptophan fluorescence measurements indicated that at pH 3.0 and 0 degrees C the pressure denaturation of trypsin is reversible but with a large hysteresis in the renaturation profile. The standard volume changes upon denaturation and renaturation are -78 mL.mol-1 and +73 mL.mol-1, respectively. However, the free energy calculated from the data in the compression and decompression directions are quite different in absolute values with + 36.6 kJ.mol-1 for the denaturation and -5 kJ. mol-1 for the renaturation. For the pressure denaturation at pH 7.3 the tryptophan fluorescence measurement and enzymatic activity assays indicated that the pressure denaturation of trypsin is irreversible. Interestingly, the study on 8-anilinonaphthalene-1-sulfonate (ANS) binding to trypsin under pressure leads to the opposite conclusion that the denaturation is reversible. FTIR spectroscopy was used to follow the changes in secondary structure. The pressure stability data found by fluorescence measurements are confirmed but the denaturation was irreversible at low and high pH in the FTIR investigation. These findings confirm that the trypsin molecule has two domains: one is related to the enzyme active site and the tryptophan residues; the other is related to the ANS binding. This is in agreement with the study on urea unfolding of trypsin and the knowledge of the molecular structure of trypsin.     

Folding studies of the cysteine proteinase inhibitor--human stefin A

Reversible GuHCl denaturation of human stefin A (25 degrees C, pH 8) was monitored by the tyrosine fluorescence, by circular dichroism in the near UV and by circular dichroism in the far UV. In each case a midpoint of 2.8 +/- 0.1 M GuHCl was obtained, demonstrating the cooperativity of the denaturation. Kinetics of the slow folding on diluting the protein from the GuHCl denatured state, was also measured by the three spectroscopic probes (10 degrees C, pH 8). Results conform to a sequential mechanism. Denaturant concentration and temperature dependence of the slow folding were measured by fluorescence. From a linear Arrhenius plot the Ea of 100 +/- 5 kJ/mol was read. 'Double mixing' experiments revealed a slow reaction going on in the unfolded state which influenced the amplitude of the fluorescence changes. 'Double mixing' experiments performed by FPLC have shown that the folding itself, i.e., the formation of a compact state, was not dependent on the time spent under unfolding conditions.     

Thermodynamic analysis of heparin binding to human antithrombin.

The binding of heparin to human antithrombin III (ATIII) was investigated by titration calorimetry (TC) and differential scanning calorimetry (DSC). TC measurements of homogeneous high-affinity pentasaccharide and octasaccharide fragments of heparin in 0.02 M phosphate buffer and 0.15 M sodium chloride (pH 7.3) yielded binding constants of (7.1 +/- 1.3) x 10(5) M-1 and (6.7 +/- 1.2) x 10(6) M-1, respectively, and corresponding binding enthalpies of -48.3 +/- 0.7 and -54.4 +/- 5.4 kJ mol-1. The binding enthalpy of heparin in phosphate buffer (0.02 M, 0.15 M NaCl, pH 7.3) was estimated from TC measurements to be -55 +/- 10 kJ mol-1, while the enthalpy in Tris buffer (0.02 M, 0.15 M NaCl, pH 7.3) was -18 +/- 2 kJ mol-1. The heparin-binding affinity was shown by fluorescence measurements not to change under these conditions. The 3-fold lower binding enthalpy in Tris can be attributed to the transfer of a proton from the buffer to the heparin-ATIII complex. DSC measurements of the ATIII unfolding transition exhibited a sharp denaturation peak at 329 +/- 1 K with a van 't Hoff enthalpy of 951 +/- 89 kJ mol-1, based on a two-state transition model and a much broader transition from 333 to 366 K. The transition peak at 329 K accounted for 9-18% of the total ATIII. At sub-saturate heparin concentrations, the lower temperature peak became bimodal with the appearance of a second transition peak at 336 K. At saturate heparin concentration only the 336 K peak was observed. This supports a two domain model of ATIII folding in which the lower stability domain (329 K) binds and is stabilized by heparin.     

Folding of dihydrofolate reductase from Escherichia coli

The urea-induced equilibrium unfolding transition of dihydrofolate reductase from Escherichia coli was monitored by UV difference, circular dichroism (CD), and fluorescence spectroscopy. Each of these data sets were well described by a two-state unfolding model involving only native and unfolded forms. The free energy of folding in the absence of urea at pH 7.8, 15 degrees C is 6.13 +/- 0.36 kcal mol-1 by difference UV, 5.32 +/- 0.67 kcal mol-1 by CD, and 5.42 +/- 1.04 kcal mol-1 by fluorescence spectroscopy. The midpoints for the difference UV, CD, and fluorescence transitions are 3.12, 3.08, and 3.18 M urea, respectively. The near-coincidence of the unfolding transitions monitored by these three techniques also supports the assignment of a two-state model for the equilibrium results. Kinetic studies of the unfolding and refolding reactions show that the process is complex and therefore that additional species must be present. Unfolding jumps in the absence of potassium chloride revealed two slow phases which account for all of the amplitude predicted by equilibrium experiments. Unfolding in the presence of 400 mM KCl results in the selective loss of the slower phase, implying that there are two native forms present in equilibrium prior to unfolding. Five reactions were observed in refolding: two slow phases designated tau 1 and tau 2 that correspond to the slow phases in unfolding and three faster reactions designated tau 3, tau 4, and tau 5 that were followed by stopped-flow techniques. The kinetics of the recovery of the native form was monitored by following the binding of methotrexate, a tight-binding inhibitor of dihydrofolate reductase, at 380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of cytidine 3'-monophosphate and uridine 3'-monophosphate with ribonuclease a at the denaturation temperature

Differential scanning calorimetry (DSC) measurements were performed on the thermal denaturation of ribonuclease a and ribonuclease a complexed with an inhibitor, cytidine or uridine 3'-monophosphate, in sodium acetate buffered solutions. Thermal denaturation of the complex results in dissociation of the complex into denatured ribonuclease a and free inhibitor. Binding constants of the inhibitor to ribonuclease a were determined from the increase in the denaturation temperature of ribonuclease a in the complexed form and from the denaturation enthalpy of the complex. Binding enthalpies of the inhibitor to ribonuclease a were determined from the increase in the denaturation enthalpy of ribonuclease a complexed with the inhibitor. For the cytidine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 87 +/- 8 M-1 (pH 7.0) to 1410 +/- 54 M-1 (pH 5.0), while the binding enthalpies increase from 17 +/- 13 kJ mol-1 (pH 4.7) to 79 +/- 15 kJ mol-1 (pH 5.5). For the uridine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 104 +/- 1 M-1 (pH 7.0) to 402 +/- 7 M-1 (pH 5.5), while the binding enthalpies increase from 16 +/- 5 kJ mol-1 (pH 6.0) to 37 +/- 4 kJ mol-1 (pH 7.0). The binding constants and enthalpies of the cytidine inhibitor in 0.05 M sodium acetate buffered solutions increase respectively from 328 +/- 37 M-1 (pH 6.5) to 2200 +/- 364 M-1 (pH 5.5) and from 22 kJ mol-1 (pH 5.5) to 45 +/- 7 kJ mol-1 (pH 6.5). the denaturation transition cooperativities of the uncomplexed and complexed ribonuclease a were close to unity, indicating that the transition is two state with a stoichiometry of 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal denaturations of staphylococcal nuclease wild-type and mutants monitored by fluorescence and circular dichroism are similar: lack of evidence for other than a two state thermal denaturation

It is unclear whether the thermal denaturation of staphylococcal nuclease is a two state, three state, or variable two state process. The thermal denaturation of wild-type staphylococcal nuclease was followed by tryptophan fluorescence and circular dichroism signal at 222 nm, forty-two and fourteen times, respectively. Analysis of this data using a simple two state model gave melting temperatures of 53.0+/-0.4 degrees C (fluorescence) and 52.7+/-0.6 degrees C (CD) and van't Hoff enthalpies of 82.4+/-2.6 kcal/mol and 88.6+/-4.2 kcal/mol. Ninety-seven mutants also had these parameters determined by both fluorescence and CD. The average difference between the melting temperatures was 1.05+/-0.75 degrees and the average difference between van't Hoff enthalpies was 1.6+/-4.8 kcal/mol. These very similar results for the two spectroscopic probes of structure are discussed in the context of the different models that have been proposed for nuclease denaturation. It is concluded, for most nuclease variants, that the errors introduced by a two state assumption are negligible and either virtually all helical structure is lost in any initial unfolding event or any intermediate must have low stability.     

Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability?

Azurin has a beta-barrel fold comprising eight beta-strands and one alpha helix. A disulfide bond between residues 3 and 26 connects the N-termini of beta strands beta1 and beta3. Three mutant proteins lacking the disulfide bond were constructed, C3A/C26A, C3A/C26I and a putative salt bridge (SB) in the C3A/S25R/C26A/K27R mutant. All three mutants exhibit spectroscopic properties similar to the wild-type protein. Furthermore, the crystal structure of the C3A/C26A mutant was determined at 2.0 A resolution and, in comparison to the wild-type protein, the only differences are found in the immediate proximity of the mutation. The mutants lose the 628 nm charge-transfer band at a temperature 10-22 degrees C lower than the wild-type protein. The folding of the zinc loaded C3A/C26A mutant was studied by guanidine hydrochloride (GdnHCl) induced denaturation monitored both by fluorescence and CD spectroscopy. The midpoint in the folding equilibrium, at 1.3 M GdnHCl, was observed using both CD and fluorescence spectroscopy. The free energy of folding determined from CD is -24.9 kJ.mol-1, a destabilization of approximately 20 kJ.mol-1 compared to the wild-type Zn2+-protein carrying an intact disulfide bond, indicating that the disulfide bond is important for giving azurin its stable structure. The C3A/C26I mutant is more stable and the SB mutant is less stable than C3A/C26A, both in terms of folding energy and thermal denaturation. The folding intermediate of the wild-type Zn2+-azurin is not observed for the disulfide-deficient C3A/C26A mutant. The rate of unfolding for the C3A/C26A mutant is similar to that of the wild-type protein, suggesting that the site of the mutation is not involved in an early unfolding reaction.     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.     

Thermodynamic stability of porcine beta-lactoglobulin. A structural relevance.

The proposed biological function of beta-lactoglobulins as transporting proteins assumes a binding ability for ligands and high stability under the acidic conditions of the stomach. This work shows that the conformational stability of nonruminant porcine beta-lactoglobulin (BLG) is not consistent with this hypothesis. Thermal denaturation of porcine BLG was studied by high-sensitivity differential scanning calorimetry within the pH range 2.0-10.0. Dependences of the denaturation temperature and enthalpy on pH were obtained, which reveal a substantial decrease in both parameters in acidic and basic media. The denaturation enthalpy follows a linear dependence on the denaturation temperature. The slope of this line is 9.4 +/- 0.6 kJ.mol-1. K-1,which is close to the denaturation heat capacity increment DeltadCp = 9.6 +/- 0.5 kJ.mol-1.K-1, determined directly from the thermograms. At pH 6.25 the denaturation temperatures of porcine and bovine BLG coincide, at 83.2 degrees C. At this pH the denaturation enthalpy of porcine BLG is 300 kJ.mol-1. The denaturation transition of porcine BLG was shown to be reversible at pH 3.0 and pH 9.0. The transition profile at both pH values follows the two-state model of denaturation. Based on the pH-dependence of the transition temperature and the linear temperature dependence of the transition enthalpy, the excess free energy of denaturation, DeltadGE, of porcine BLG was calculated as a function of pH and compared with that of bovine BLG derived from previously reported data. The pH-dependence of DeltadGE is analysed in terms of the contributions of side-chain H-bonds to the protein stability. Interactions stabilizing native folds of porcine and bovine BLG are discussed.     

DSC studies of the conformational stability of barstar wild-type.

The temperature induced unfolding of barstar wild-type of bacillus amyloliquefaciens (90 residues) has been characterized by differential scanning microcalorimetry. The process has been found to be reversible in the pH range from 6.4 to 8.3 in the absence of oxygen. It has been clearly shown by a ratio of delta HvH/delta Hcal near 1 that denaturation follows a two-state mechanism. For comparison, the C82A mutant was also studied. This mutant exhibits similar reversibility, but has a slightly lower transition temperature. The transition enthalpy of barstar wt (303 kJ mol-1) exceeds that of the C82A mutant (276 kJ mol-1) by approximately 10%. The heat capacity changes show a similar difference, delta Cp being 5.3 +/- 1 kJ mol-1 K-1 for the wild-type and 3.6 +/- 1 kJ mol-1 K-1 for the C82A mutant. The extrapolated stability parameters at 25 degrees C are delta G0 = 23.5 +/- 2 kJ mol-1 for barstar wt and delta G0 = 25.5 +/- 2 kJ mol-1 for the C82A mutant.     

Thermodynamics of the denaturation of pepsinogen by urea.

The denaturation of swine pepsinogen has been studied as a function of urea concentration, pH, and temperature. The unfolding of the protein by urea has been found to be fully reversible under different conditions of pH, temperature, and denaturant concentration. Kinetic experiments have shown that the transition shows two-state behavior at 25 degrees C in the pH range 6-8 covered in this study. Analysis of the equilibrium data obtained at 25 degrees C according to Tanford (Tanford, C. (1970), Adv. Protein Chem. 24, 1) and Pace (Pace, N.C. (1975), Crit. Rev. Biochem. 3, 1) leads to the conclusion that the free energy of stabilization of native pepsinogen, relative to the denatured state, under physiological conditions, is only 6-12 kcal mol-1. The temperature dependence of the equilibrium constant for the unfolding of pepsinogen by urea in the range 20-50 degrees C at pH 8.0 can be described by assigning the following values of thermodynamic parameters for the denaturation at 25 degrees C: deltaH=31.5 kcal mol-1; deltaS=105 cal deg-1 mol-1; and deltaCp=5215 cal deg-1 mol-1.     

Structural and thermodynamic studies of KM+, a d-mannose binding lectin from Artocarpus integrifolia seeds

The KM+ lectin exhibits a novel and unusual circular dichroism (CD) spectrum that could be explained by a high proline content that would be inducing deformation of the beta-structure and/or unusual turns. KM+ was shown to be a very rigid lectin, which was very stable under a broad variety of conditions (urea, guanidine, hydrolysis, pH, etc.). Only incubation for 60 min at 333-338 K and extreme basic pH were able to induce conformational changes which could be observed by CD and fluorescence measurements. Data from CD are typical for protein denaturing associated with changes in the overall secondary structure. Data from high-performance size exclusion chromatography (SEC) showed that the denatured forms produced at pH 12.0 are eluted in clusters that co-elute with the native forms. A significant contribution from the tyrosines to the fluorescence emission upon denaturation was observed above 328 K. In fact at 328 K some broadening of the emission spectrum takes place followed by the appearance of a shoulder (approx. 305 nm) at 333 K and above. The sensitivity of tryptophan fluorescence to the addition of sugar suggests a close proximity of the tryptophan residues to the sugar binding site, K(a)=(2.9+/-0.6)x10(3) M(-1). The fraction of chromophore accessible to the quencher obtained is f(a)=0.43+/-0.08, suggesting that approximately 50% of the tryptophan residues are not accessible to quenching by d-mannose. KM+ thermal denaturation was found to be irreversible and was analyzed using a two-state model (N-->D). The results obtained for the activation energy and transition temperature from the equilibrium CD studies were: activation energy, E(a)=134+/-11 kJ/mol and transition temperature, T(m)=339+/-1 K, and from the fluorescence data: E(a)=179+/-18 kJ/mol and T(m)=337+/-1 K. Kinetic studies gave the following values: E(a)=108+/-18 kJ/mol and E(a)=167+/-12 kJ/mol for CD and fluorescence data, respectively.     

Reactivation and refolding of rabbit muscle creatine kinase denatured in 2,2,2-trifluoroethanol solutions

The unfolding and refolding of creatine kinase (ATP:creatine N-phosphotransferase (CK), EC 2.7.3.2) during denaturation and reactivation by trifluoroethanol (TFE) have been studied. Significant aggregation was observed when CK was denatured at TFE concentrations between 10% and 40% (v/v). 50% TFE (v/v) was used to study the denaturation and unfolding of CK. The activity loss of CK was a very quick process, as was the marked conformational changes during denaturation followed by fluorescence emission spectra and far-ultraviolet CD spectra. DTNB modification and size exclusion chromatography were used to find that CK dissociated and was in its monomer state after denaturation with 50% TFE. Reactivation and refolding were observed after 80-fold dilution of the denatured CK into 0.05 M Tris-HCl buffer, pH 8.0. The denatured CK recovered about 38% activity following a two phase course (k(1)=4.82+/-0.41x10(-3) s(-1), k(2)=0.60+/-0.01x10(-3) s(-1)). Intrinsic fluorescence maximum intensity changes showed that the refolding process also followed biphasic kinetics (k(1)=4.34+/-0.27x10(-3) s(-1), k(2)=0.76+/-0.02x10(-3) s(-1)) after dilution into the proper solutions. The far-ultraviolet CD spectra ellipticity changes at 222 nm during the refolding process also showed a two phase course (k(1)=4.50+/-0.07x10(-3) s(-1), k(2)=1.13+/-0.05x10(-3) s(-1)). Our results suggest that TFE can be used as a reversible denaturant like urea and GuHCl. The 50% TFE induced CK denaturation state, which was referred to as the 'TFE state', and the partially refolded CK are compared with the molten globule state. The aggregation caused by TFE during denaturation is also discussed in this paper.     

Unfolding of the trp repressor from Escherichia coli monitored by fluorescence, circular dichroism and nuclear magnetic resonance

The denaturation of the trp repressor from Escherichia coli has been studied by fluorescence, circular dichroism and proton magnetic resonance spectroscopy. The dependences of the fluorescence emission of the two tryptophan residues on the concentration of urea are not identical. The dependence of the quenching of tryptophan fluorescence by iodide as a function of urea concentration also rules out a two-state transition. The circular dichroism at 222 nm decreases in two phases as urea is added. Normalised curves for different residues observed by 1H NMR also do not coincide, and require the presence of at least one stable intermediate. Analysis of the dependence of the denaturation curves on the concentration of protein indicate that the first transition is a partial unfolding of the dimeric repressor, resulting in a loss of about 25% of the helical content. The second transition is the dissociation and unfolding of the partially unfolded dimer. At high concentrations of protein (500 microM) about 73% of the repressor exists as the intermediate in 4 M urea. The apparent dissociation constant is about 10(-4) M; the subunits are probably strongly stabilised by the subunit interaction. The native repressor is stable up to at least 70 degrees C, whereas the intermediate formed at 4 M urea can be denatured reversibly by heating (melting temperature approximately 60 degrees C, delta H approximately 230 kJ/mol).     

Thermodynamic analysis of the structural stability of phage 434 Cro protein

Thermodynamic parameters describing the phage 434 Cro protein have been determined by calorimetry and, independently, by far-UV circular dichroism (CD) measurements of isothermal urea denaturations and thermal denaturations at fixed urea concentrations. These equilibrium unfolding transitions are adequately described by the two-state model. The far-UV CD denaturation data yield average temperature-independent values of 0.99 +/- 0.10 kcal mol(-)(1) M(-)(1) for m and 0.98 +/- 0.05 kcal mol(-)(1) K(-)(1) for DeltaC(p)()(,U), the heat capacity change accompanying unfolding. Calorimetric data yield a temperature-independent DeltaC(p)()(,U) of 0.95 +/- 0.30 kcal mol(-)(1) K(-)(1) or a temperature-dependent value of 1.00 +/- 0.10 kcal mol(-)(1) K(-)(1) at 25 degrees C. DeltaC(p)()(,U) and m determined for 434 Cro are in accord with values predicted using known empirical correlations with structure. The free energy of unfolding is pH-dependent, and the protein is completely unfolded at pH 2.0 and 25 degrees C as judged by calorimetry or CD. The stability of 434 Cro is lower than those observed for the structurally similar N-terminal domain of the repressor of phage 434 (R1-69) or of phage lambda (lambda(6)(-)(85)), but is close to the value reported for the putative monomeric lambda Cro. Since a protein's structural stability is important in determining its intracellular stability and turnover, the stability of Cro relative to the repressor could be a key component of the regulatory circuit controlling the levels and, consequently, the functions of the two proteins in vivo.     

Equilibrium studies of the effect of difference in sequence homology on the mechanism of denaturation of bovine and horse cytochromes-c

We have carried out equilibrium studies of the effect of the amino acid residue difference in the primary structure of bovine cytochrome-c (b-cyt-c) and horse cyt-c (h-cyt-c) on the mechanism of their folding <--> unfolding processes at pH 6.0 and 25 degrees C. It has been observed that guanidinium chloride (GdmCl)-induced denaturation of b-cyt-c follows a two-state mechanism and that of h-cyt-c is not a two-state process. This conclusion is reached from the coincidence and non-coincidence of GdmCl-induced transition curves of bovine and horse proteins, respectively, monitored by measurements of absorbance at 405, 530 and 695 nm and circular dichroism (CD) at 222, 416 and 405 nm. These measurements on h-cyt-c in the presence of GdmCl in the concentration range 0.75-2.0 M also suggest that the protein retains all the native far-UV CD but has slightly perturbed tertiary interaction. The intermediate in the presence of these low denaturant concentrations does not have the structural characteristics of a molten globule as judged by the 8-Anilino-1-napthalene sulfonic acid (ANS) binding and near-UV CD experiments. We have also carried out thermal denaturation studies of bovine and horse cyts-c in the presence of GdmCl monitored by absorbance at 405 nm and far-UV CD at 222 nm. The heat-induced denaturation measurements in the presence of the denaturant show (1) that denaturation of b-cyt-c is a two-state process and that of h-cyt-c does not follow a two-state mechanism, and (2) that the enthalpy change on denaturation of both proteins strongly depends on GdmCl concentration.     

Equilibrium and kinetic studies on reversible and irreversible denaturation of micrococcal nuclease

The effect of pH and temperature on the thermal denaturation of micrococcal nuclease wer4e investigated. The ranges employed were between pH3.30 and pH9.70 and between 10 degrees C and 85 degrees C, respectively. The reversible denaturation involved in the whole process was clearly discriminated from the irreversible one. The former took place with a large enthalpy change of 384 kJ mol(-1) at pH 9.70, where the enzyme exhibited it s maximum activity. The latter probably led to aggregation because the successive long incubation after complete deactivation caused precipitation. A reasonable scheme explaining the process involving both denaturations was proposed and the kinetic on the irreversible deactivation was performed. It was revealed that the irreversible deactivation involved two types of reactions whose activation energies were relatively small: 22.2 kJ mol(-1) and 18.8kJ mol(-1). The presence of sucrose suppressed the reversible denaturation without significant influence on enthalpy change, whereas it affected little the irreversible deactivation kinetically. The effects of pH change and addition of sucrose on the denaturation were discussed thermodynamically, especially in terms of the entropy change. (c) 1994 John Wiley & Sons, Inc.