Differential scanning calorimetric,circular dichroism,and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans

The thermal unfolding of xylanase A from Streptomyces lividans, and of its isolated substrate binding and catalytic domains, was studied by differential scanning calorimetry and Fourier transform infrared and circular dichroism spectroscopy. Our calorimetric studies show that the thermal denaturation of the intact enzyme is a complex process consisting of two endothermic events centered near 57 and 64 degrees C and an exothermic event centered near 75 degrees C, all of which overlap slightly on the temperature scale. A comparison of the data obtained with the intact enzyme and isolated substrate binding and catalytic domains indicate that the lower- and higher-temperature endothermic events are attributable to the thermal unfolding of the xylan binding and catalytic domains, respectively, whereas the higher-temperature exothermic event arises from the aggregation and precipitation of the denatured catalytic domain. Moreover, the thermal unfolding of the two domains of the native enzyme are thermodynamically independent and differentially sensitive to pH. The unfolding of the substrate binding domain is a reversible two-state process and, under appropriate conditions, the refolding of this domain to its native conformation can occur. In contrast, the unfolding of the catalytic domain is a more complex process in which two subdomains unfold independently over a similar temperature range. Also, the unfolding of the catalytic domain leads to aggregation and precipitation, which effectively precludes the refolding of the protein to its native conformation. These observations are compatible with the results of our spectroscopic studies, which show that the catalytic and substrate binding domains of the enzyme are structurally dissimilar and that their native conformations are unaffected by their association in the intact enzyme. Thus, the calorimetric and spectroscopic data demonstrate that the S. lividans xylanase A consists of structurally dissimilar catalytic and substrate binding domains that, although covalently linked, undergo essentially independent thermal denaturation. These observations provide valuable new insights into the structure and thermal stability of this enzyme and should assist our efforts at engineering xylanases that are more thermally robust and otherwise better suited for industrial applications.     

Linked thermal and solute perturbation analysis of cooperative domain interactions in proteins. Structural stability of diphtheria toxin

The temperature and guanidine hydrochloride (GuHCl) dependence of the structural stability of diphtheria toxin has been investigated by high-sensitivity differential scanning calorimetry. In 50 mM phosphate buffer at pH 8.0 and in the absence of GuHCl, the thermal unfolding of diphtheria toxin is characterized by a transition temperature (Tm) of 54.9 degrees C, a calorimetric enthalpy change (delta H) of 295 kcal/mol, and a van't Hoff to calorimetric enthalpy ratio of 0.57. Increasing the GuHCl concentration lowers the transition temperature and the calorimetric enthalpy change. At the same time, the van't Hoff to calorimetric enthalpy ratio increases until it reaches a value of 1 at 0.3 M GuHCl and remains constant thereafter. At low GuHCl concentrations (0-0.3 M), the thermal unfolding of diphtheria toxin is characterized by the presence of two transitions corresponding to the A and B domains of the protein. At higher GuHCl concentrations (0.3-1 M), the A domain is unfolded at all temperatures, and only one transition corresponding to the B domain is observed. Under these conditions, the most stable protein conformation at low temperatures is a partially folded state in which the A domain is unfolded and the B domain folded. A general model that explicitly considers the energetics of domain interactions has been developed in order to account for the stability and cooperative behavior of diphtheria toxin. It is shown that this cooperative domain interaction model correctly accounts for the temperature location as well as the shape and area of the calorimetric curves. Under physiological conditions, domain-domain interactions account for most of the structural stability of the A domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phage P22 tailspike protein: removal of head-binding domain unmasks effects of folding mutations on native-state thermal stability.

A shortened, recombinant protein comprising residues 109-666 of the tailspike endorhamnosidase of Salmonella phage P22 was purified from Escherichia coli and crystallized. Like the full-length tailspike, the protein lacking the amino-terminal head-binding domain is an SDS-resistant, thermostable trimer. Its fluorescence and circular dichroism spectra indicate native structure. Oligosaccharide binding and endoglycosidase activities of both proteins are identical. A number of tailspike folding mutants have been obtained previously in a genetic approach to protein folding. Two temperature-sensitive-folding (tsf) mutations and the four known global second-site suppressor (su) mutations were introduced into the shortened protein and found to reduce or increase folding yields at high temperature. The mutational effects on folding yields and subunit folding kinetics parallel those observed with the full-length protein. They mirror the in vivo phenotypes and are consistent with the substitutions altering the stability of thermolabile folding intermediates. Because full-length and shortened tailspikes aggregate upon thermal denaturation, and their denaturant-induced unfolding displays hysteresis, kinetics of thermal unfolding were measured to assess the stability of the native proteins. Unfolding of the shortened wild-type protein in the presence of 2% SDS at 71 degrees C occurs at a rate of 9.2 x 10(-4) s(-1). It reflects the second kinetic phase of unfolding of the full-length protein. All six mutations were found to affect the thermal stability of the native protein. Both tsf mutations accelerate thermal unfolding about 10-fold. Two of the su mutations retard thermal unfolding up to 5-fold, while the remaining two mutations accelerate unfolding up to 5-fold. The mutational effects can be rationalized on the background of the recently determined crystal structure of the protein.     

Thermal unfolding studies of a phytocyanin

The thermal stability of umecyanin, a stellacyanin from horseradish roots, has been investigated by differential scanning calorimetry, optical absorption and fluorescence spectroscopy at neutral and alkaline pH. Above pH 9 the Cu(II) protein experiences a blue shift of the main visible absorption band at approximately 600 nm and changes colour from blue to violet. The thermal transition of the protein is irreversible and occurs between 61.4 and 68.8 degrees C at pH 7.5 and between 50.7 and 57.4 degrees C at pH 9.8. The calorimetric data indicates that at both pH values the thermally induced transition of the protein between the native and denaturated states can be described in terms of the classical Lumry-Eyring unfolding model Native<-->Unfolded-->Final. The analysis of the reversible step in the unfolding pathway demonstrates a significant reduction in conformational stability (DeltaG) of the alkaline form of the protein. Such a reduction is consistent with an enhanced flexibility of UMC at high pH and has mainly entropic character.     

Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance.

Streptococcus equisimilis streptokinase (SK) is a single-chain protein of 414 residues that is used extensively in the clinical treatment of acute myocardial infarction due to its ability to activate human plasminogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and their complex. We reported recently (Parrado J et al., 1996, Protein Sci 5:693-704) that SK is composed of three structural domains (A, B, and C) with a C-terminal tail that is relatively unstructured. Here, we report thermal unfolding experiments, monitored by CD and NMR, using samples of intact SK, five isolated SK fragments, and two two-chain noncovalent complexes between complementary fragments of the protein. These experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and interdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individual domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximately 46 degrees C and its stability is largely independent of the presence of the other domains. The high-temperature transition in intact SK (at approximately 63 degrees C) corresponds to the unfolding of both domains A and C. Thermal stability of domain C is significantly increased by its isolation from the rest of the chain. By contrast, cleavage of the Phe 63-Ala 64 peptide bond within domain A causes thermal destabilization of this domain. The two resulting domain portions (A1 and A2) adopt unstructured conformations when separated. A1 binds with high affinity to all fragments that contain the A2 portion, with a concomitant restoration of the native-like fold of domain A. This result demonstrates that the mechanism whereby A1 stimulates the plasminogen activator activities of complementary SK fragments is the reconstitution of the native-like structure of domain A.     

Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme.The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.     

Equilibrium heat-induced denaturation of chitinase 40 from Streptomyces thermoviolaceus

High-precision differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to study the thermal unfolding of chitinase 40 (Chi40) from Streptomyces thermoviolaceus. Chi40 belongs to family 18 of glycosyl hydrolase superfamily bearing a catalytic domain with a "TIM barrel"-like fold, which exhibits deviations from the (beta/alpha)8 fold. The thermal unfolding is reversible at pH = 8.0 and 9.0. The denatured state is characterized by extensive structural changes with respect to the native. The process is characterized by slow relaxation kinetics. Even slower refolding rates are recorded upon cooling. It is shown that the denaturation calorimetric data obtained at slow heating rate (0.17 K/min) are in excellent agreement with equilibrium data obtained by extrapolation of the experimental results to zero scanning rate. Analysis of the DSC results reveals that the experimental data can be successfully fitted using either a non-two-state sequential model involving one equilibrium intermediate, or an independent transitions model involving the unfolding of two Chi40 energetic domains to intermediate states. The stability of the native state with respect to the final denatured state is estimated, deltaG = 24.0 kcal/mol at 25 degrees C. The thermal results are in agreement with previous findings from chemical denaturation studies of a wide variety of (beta/alpha)8 barrel proteins, that their unfolding is a non-two-state process, always involving at least one unfolding intermediate.     

A comparative investigation of the thermal unfolding of pseudoazurin in the Cu(II)-holo and apo form

The contribution of the copper ion to the stability and to the unfolding pathway of pseudoazurin was investigated by a comparative analysis of the thermal unfolding of the Cu(II)-holo and apo form of the protein. The unfolding has been followed by calorimetry, fluorescence, optical density, and electron paramagnetic resonance (EPR) spectroscopy. The thermal transition of Cu(II)-holo pseudoazurin is irreversible and occurs between 60.0 and 67.3 degrees C, depending on the scan rate and technique used. The denaturation pathway of Cu(II)-holo pseudoazurin can be described by the Lumry-Eyring model: N --> U --> [corrected] F; the protein reversibly goes from the native (N) to the unfolded (U) state, and then irreversibly to the final (F) state. The simulation of the experimental calorimetric profiles, according to this model, allowed us to determine the thermodynamic and kinetic parameters of the two steps. The DeltaG value calculated for the Cu(II)-holo pseudoazurin is 39.2 kJ.mol(-1) at 25 degrees C. The sequence of events in the denaturation process of Cu(II)-holo pseudoazurin emergence starts with the disruption of the copper site and the hydrophobic core destabilization followed by the global protein unfolding. According to the EPR findings, the native type-1 copper ion shows type-2 copper features after the denaturation. The removal of the copper ion (apo form) significantly reduces the stability of the protein as evidenced by a DeltaG value of 16.5 kJ.mol(-1) at 25 degrees C. Moreover, the apo Paz unfolding occurs at 41.8 degrees C and is compatible with a two-state reversible process N --> [corrected] U.     

Molecular evolution of the thermosensitive PAb1620 epitope of human p53 by DNA shuffling.

Conformational stability of the p53 protein is an absolute necessity for its physiological function as a tumor suppressor. Recent in vitro studies have shown that wild-type p53 is a highly temperature-sensitive protein at the structural and functional levels. Upon heat treatment at 37 degrees C, p53 loses its wild-type (PAb1620(+)) conformation and its ability to bind DNA, but can be stabilized by different classes of ligands. To further investigate the thermal instability of p53, we isolated p53 mutants resistant to heat denaturation. For this purpose, we applied a recently developed random mutagenesis technique called DNA shuffling and screened for p53 variants that could retain reactivity to the native conformation-specific anti-p53 antibody PAb1620 upon thermal treatment. After three rounds of mutagenesis and screening, mutants were isolated with the desired phenotype. The isolated mutants were translated in vitro in either Escherichia coli or rabbit reticulocyte lysate and characterized biochemically. Mutational analysis identified 20 amino acid residues in the core domain of p53 (amino acids 101-120) responsible for the thermostable phenotype. Furthermore, the thermostable mutants could partially protect the PAb1620(+) conformation of tumor-derived p53 mutants from thermal unfolding, providing a novel approach for restoration of wild-type structure and possibly function to a subset of p53 mutants in tumor cells.     

Stability of bacteriophage T4 short tail fiber

Adsorption of T-even bacteriophages to the E. coli host cell is mediated by long and short tail fibers. Bacteriophage T4 short tail fiber protein p12 was used to investigate the stability against thermal and chemical denaturation. Purified p12 is thermostable with a melting point of 78 degrees C. Guanidinium chloride-induced denaturation displayed strong hysteresis and an intermediate between 2 and 3 M denaturant. The transitions occur at 1.5 and 3.2 M denaturant as revealed by fluorescence spectroscopy and circular dichroism. The data suggest an equilibrium unfolding intermediate with a separate unfolding of the C-terminal knob domain and the shaft region.     

The stability of the TIM-barrel domain of a psychrophilic chitinase

Chitinase 60 from the psychrophilic bacterium Moritella marina (MmChi60) is a four-domain protein whose structure revealed flexible hinge regions between the domains, yielding conformations in solution that range from fully extended to compact. The catalytic domain is a shallow-grooved TIM-barrel. Heat-induced denaturation experiments of the wild-type and mutants resulting from the deletions of the two-Ig-like domains and the chitin binding domain reveal calorimetric profiles that are consistent with non-collaborative thermal unfolding of the individual domains, a property that must be associated to the “hinge-regions”. The calorimetric measurements of the (β/α)₈ catalytic domain reveal that the thermal unfolding is a slow-relaxation transition exhibiting a stable, partially structured intermediate state. Circular dichroism provides evidence that the intermediate exhibits features of a molten globule i.e., loss of tertiary structure while maintaining the secondary structural elements of the native. GdnHCl-induced denaturation studies of the TIM-barrel demonstrate an extraordinarily high resistance to the denaturant. Slow-relaxation kinetics characterize the unfolding with equilibration times exceeding six days, a property that is for the first time observed for a psychrophilic TIM barrel. On the other hand, the thermodynamic stability is ΔG=6.75±1.3 kcal/mol, considerably lower than for structural-insertions-containing barrels. The mutant E153Q used for the crystallographic studies of MmChi60 complexes with NAG ligands has a much lower stability than the wild-type.     

Structural stability and unfolding properties of thermostable bacterial alpha-amylases: a comparative study of homologous enzymes

In a comparative investigation on two thermostable alpha-amylases [Bacillus amyloliquefaciens (BAA), T(m) = 86 degrees C and Bacillus licheniformis (BLA), T(m) = 101 degrees C], we studied thermal and guanidine hydrochloride (GndHCl)-induced unfolding using fluorescence and CD spectroscopy, as well as dynamic light scattering. Depletion of calcium from specific ion-binding sites in the protein structures reduces the melting temperature tremendously for both alpha-amylases. The reduction is nearly the same for both enzymes, namely, in the order of 50 degrees C. Thus, the difference in thermostability between BLA and BAA (DeltaT(m) approximately 15 degrees C) is related to intrinsic properties of the respective protein structures themselves and is not related to the strength of ion binding. The thermal unfolding of both proteins is characterized by a full disappearance of secondary structure elements and by a concurrent expansion of the 3D structure. GndHCl-induced unfolding also yields a fully vanishing secondary structure but with more expanded 3D structures. Both alpha-amylases remain much more compact upon thermal unfolding as compared to the fully unfolded state induced by chemical denaturants. Such rather compact thermal unfolded structures lower the conformational entropy change during the unfolding transition, which principally can contribute to an increased thermal stability. Structural flexibilities of both enzymes, as measured with tryptophan fluorescence quenching, are almost identical for both enzymes in the native states, as well as in the unfolded states. Furthermore, we do not observe any difference in the temperature dependence of the structural flexibilities between BLA and BAA. These results indicate that conformational dynamics on the time scale of our studies seem not to be related to thermal stability or to thermal adaptation.     

Unusual cold denaturation of a small protein domain

A thermal unfolding study of the 45-residue α-helical domain UBA(2) using circular dichroism is presented. The protein is highly thermostable and exhibits a clear cold unfolding transition with the onset near 290 K without denaturant. Cold denaturation in proteins is rarely observed in general and is quite unique among small helical protein domains. The cold unfolding was further investigated in urea solutions, and a simple thermodynamic model was used to fit all thermal and urea unfolding data. The resulting thermodynamic parameters are compared to those of other small protein domains. Possible origins of the unusual cold unfolding of UBA(2) are discussed.     

Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase

The flavoenzyme DAAO from Rhodotorula gracilis, a structural paradigm of the glutathione-reductase family of flavoproteins, is a stable homodimer with a flavin adenine dinucleotide (FAD) molecule tightly bound to each 40-kD subunit. In this work, the thermal unfolding of dimeric DAAO was compared with that of two monomeric forms of the same protein: a Deltaloop mutant, in which 14 residues belonging to a loop connecting strands betaF5-betaF6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH(4)SCN. Thiocyanate specifically and reversibly affects monomer association in wild-type DAAO by acting on hydrophobic residues and on ionic pairs between the betaF5-betaF6 loop of one monomer and the alphaI3' and alphaI3" helices of the symmetry-related monomer. By using circular dichroism spectroscopy, protein and flavin fluorescence, activity assays, and DSC, we demonstrated that thermal unfolding involves (in order of increasing temperatures) loss of tertiary structure, followed by loss of some elements of secondary structure, and by general unfolding of the protein structure that was concomitant to FAD release. Temperature stability of wild-type DAAO is related to the presence of a dimeric structure that affects the stability of independent structural domains. The monomeric Deltaloop mutant is thermodynamically less stable than dimeric wild-type DAAO (with melting temperatures (T(m)s) of 48 degrees C and 54 degrees C, respectively). The absence of complications ensuing from association equilibria in the mutant Deltaloop DAAO allowed identification of two energetic domains: a low-temperature energetic domain related to unfolding of tertiary structure, and a high-temperature energetic domain related to loss of secondary structure elements and to flavin release.     

Thermal unfolding of small proteins with SH3 domain folding pattern

The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy. The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec). At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12–16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities. Proteins 31:309–319, 1998. © 1998 Wiley-Liss, Inc.     

Unfolding by temperature and guanidine hydrochloride of chicken pancreatic polypeptide

Thermal unfolding of chicken pancreatic polypeptide at two different concentrations was studied at various pH values. The thermal stability was higher at higher protein concentrations. The transition temperatures at two different protein concentrations changed with pH in parallel and decreased by about 30 degrees C on lowering pH from 5 to 2. The results on the thermal unfolding were analyzed by assuming that the dimerization constant is independent of pH, that the thermal unfolding occurs only after the pancreatic polypeptide dimers dissociated into the monomers, and that one ionizable group participates in the acid unfolding of the monomer. The free energy change for the unfolding of the pancreatic polypeptide monomer was estimated to be 1.4 kcal/mol. The unfolding of pancreatic polypeptide by guanidine hydrochloride at pH 6.0 and 25 degrees C was also studied. The stability to guanidine hydrochloride was higher at higher protein concentrations.     

Effect of the polypeptide binding on the thermodynamic stability of the substrate binding domain of the DnaK chaperone

The effect of polypeptide binding on the stability of the substrate binding domain of the molecular chaperone DnaK has been studied by thermodynamic analysis. The calorimetric scan of the fragment of the substrate binding domain DnaK384-638, consisting of a beta-domain and an alpha-helical lid, showed two transitions centered at 56.2 and 76.0 degrees C. On the other hand, the thermal unfolding of the shorter fragment DnaK386-561, which lacks half of the alpha-helical lid, exhibited a single transition at 57.0 degrees C. Therefore, the transition of DnaK384-638 at 56.2 degrees C is mainly attributed to the unfolding of the beta-domain. The calorimetric scan of DnaK384-638D526N showed that the unfolding of the beta-domain was composed of two transitions. The polypeptide bound DnaK384-638 exhibited a symmetrical DSC peak at 58.6 degrees C, indicating that the substrate binding shifts the beta-domain toward a single cooperative unit. A low concentration of GdnHCl (<1.0 M) induced a conformational change in the beta-domain of DnaK384-638 without changes in the secondary structure. While the thermal unfolding of the beta-domain of DnaK384-638 was composed of two transitions in the presence of GdnHCl, the beta-domain of the substrate bound DnaK384-638 exhibited a single symmetrical DSC peak in the same condition. All together, our results indicate that complex between DnaK384-638 and substrate forms a rigid conformation in the beta-domain.     

Evolutionary stabilization of the gene-3-protein of phage fd reveals the principles that govern the thermodynamic stability of two-domain proteins

The gene-3-protein (G3P) of filamentous phage is essential for their propagation. It consists of three domains. The CT domain anchors G3P in the phage coat, the N2 domain binds to the F pilus of Escherichia coli and thus initiates infection, and the N1 domain continues by interacting with the TolA receptor. Phage are thus only infective when the three domains of G3P are tightly linked, and this requirement is exploited by Proside, an in vitro selection method for proteins with increased stability. In Proside, a repertoire of variants of the protein to be stabilized is inserted between the N2 and the CT domains of G3P. Stabilized variants can be selected because they resist cleavage by a protease and thus maintain the essential linkage between the domains. The method is limited by the proteolytic stability of G3P itself. We improved the stability of G3P by subjecting the phage without a guest protein to rounds of random in vivo mutagenesis and proteolytic Proside selections. Variants of G3P with one to four mutations were selected, and the temperature at which the corresponding phage became accessible for a protease increased in a stepwise manner from 40 degrees C to almost 60 degrees C. The N1-N2 fragments of wild-type gene-3-protein and of the four selected variants were purified and their stabilities towards thermal and denaturant-induced unfolding were determined. In the biphasic transitions of these proteins domain dissociation and unfolding of N2 occur in a concerted reaction in the first step, followed by the independent unfolding of domain N1 in the second step. N2 is thus less stable than N1, and it unfolds when the interactions with N1 are broken. The strongest stabilizations were caused by mutations in domain N2, in particular in its hinge subdomain, which provides many stabilizing interactions between the N1 and N2 domains. These results reveal how the individual domains and their assembly contribute to the overall stability of two-domain proteins and how mutations are optimally placed to improve the stability of such proteins.     

Thermal unfolding of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase studied by differential scanning calorimetry.

Thermal unfolding parameters were determined for a two-domain tetrameric enzyme, phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and for its isolated NAD(+)-binding domain. At pH 8.0, the transition temperatures (t(max)) for the apoforms of the native Bacillus stearothermophilus GAPDH and the isolated domain were 78.3 degrees C and 61.9 degrees C, with calorimetric enthalpies (DeltaH(cal)) of 4415 and 437 kJ/mol (or 30.7 and 22.1 J/g), respectively. In the presence of nearly saturating NAD(+) concentrations, the t(max) and the DeltaH(cal) increased by 13.6 degrees C and by 2365 kJ/mol, respectively, for the native apoenzyme, and by 2.8 degrees C and 109 kJ/mol for the isolated domain. These results indicate that interdomain interactions are essential for NAD(+) to produce its stabilizing effect on the structure of the native enzyme. The thermal stability of the isolated NAD(+)-binding domain increased considerably upon transition from pH 6.0 to 8.0. By contrast, native GAPDH exhibited greater stability at pH 6.0; similar pH-dependencies of thermal stability were displayed by GAPDHs isolated from rabbit muscle and Escherichia coli. The binding of NAD(+) to rabbit muscle apoenzyme increased t(max) and DeltaH(cal) and diminished the widths of the DSC curves; the effect was found to grow progressively with increasing coenzyme concentrations. Alkylation of the essential Cys149 with iodoacetamide destabilized the apoenzyme and altered the effect of NAD(+). Replacement of Cys149 by Ser or by Ala in the B. stearothermophilus GAPDH produced some stabilization, the effect of added NAD(+) being basically similar to that observed with the wild-type enzyme. These data indicate that neither the ion pairing between Cys149 and His176 nor the charge transfer interaction between Cys149 and NAD(+) make any significant contribution to the stabilization of the enzyme's native tertiary structure and the accomplishment of NAD(+)-induced conformational changes. The H176N mutant exhibited dramatically lower heat stability, as reflected in the values of both DeltaH(cal) and t(max). Interestingly, NAD(+) binding resulted in much wider heat capacity curves, suggesting diminished cooperativity of the unfolding transition.     

Unfolding domains of recombinant fusion alpha alpha-tropomyosin.

The thermal unfolding of the coiled-coil alpha-helix of recombinant alpha alpha-tropomyosin from rat striated muscle containing an additional 80-residue peptide of influenza virus NS1 protein at the N-terminus (fusion-tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion-tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 degrees C involving the middle of the molecule; (2) a major transition at 46 degrees C involving no more than 36% of the helix from the C-terminus; (3) a major transition at 56 degrees C involving about 46% of the helix from the N-terminus; and (4) a transition from the nonhelical fusion domain at about 70 degrees C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 degrees C or 70 degrees C transition. The very stable fusion unfolding domain of fusion-tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a cross-link to stabilize the adjacent N-terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C-terminal domain at 46 degrees C from the stabilized N-terminal unfolding domain at 56 degrees C. Thus, strong localized interchain interactions in coiled-coil molecules can increase the stability of neighboring domains.