Stability parameters for one-step mechanism of irreversible protein denaturation: a method based on nonlinear regression of calorimetric peaks with nonzero deltaCp

Thermal transitions of many proteins have been found to be calorimetrically irreversible and scan-rate dependent. Calorimetric determinations of stability parameters of proteins which unfold irreversibly according to a first-order kinetic scheme have been reported. These methods require the approximation that the increase in heat capacity upon denaturation deltaCp is zero. A method to obtain thermodynamic parameters and activation energy for the two-state irreversible process N --> D from nonlinear fitting to calorimetric traces is proposed here. It is based on a molar excess heat capacity function which considers irreversibility and a nonzero constant deltaCp. This function has four parameters: (1) temperature at which the calorimetric profile reaches its maximal value (Tm), (2) calorimetric enthalpy at Tm (deltaHm), (3) deltaCp, and (4) activation energy (E). The thermal irreversible denaturation of subtilisin BPN' from Bacillus amyloliquefaciens was studied by differential scanning calorimetry at pH 7.5 to test our model. Transitions were found to be strongly scanning-rate dependent with a mean deltaCp value of 5.7 kcal K(-1)mol(-1), in agreement with values estimated by accessible surface area and significantly higher than a previously reported value.     

Effect of D-amino acids on the functional activity and conformational stability of ribonuclease-A

Using cytidine 2':3' cyclic monophosphate as a substrate, Km and k(cat) of ribonuclease-A in the presence of different concentrations of D-amino acids (Ala, Ser, Pro and Lys) and their L-isomers were measured at pH 6.0 and 25 degrees C. These kinetic parameters remained unchanged in the presence and absence of D-and L-amino acids. This is the first experimental evidence showing that D-amino acids are compatible with the enzyme function. Values of Tm (midpoint of denaturation), deltaHm (enthalpy change at Tm) and deltaCp (constant-pressure heat capacity change) were also determined from the heat-induced denaturation curves of the protein, measured in the presence and absence of D- and L-isomers of an amino acid at four different pH values. It is shown for the first time that these thermodynamic parameters, within experimental errors, do not depend on the stereospecificity of an amino acid. Estimates of deltaGDo with the help of Gibbs-Helmoltz equation (deltaGDo = deltaHm (1-298.15/Tm)--deltaCp [(Tm-298.15) + 298.15 In (298.15/Tm)]) using known values of Tm, deltaHm and deltaCp suggested that D- and L-amino acids are compatible with protein stability, for deltaGDo remained unchanged in the presence of amino acids.     

Low molecular mass pectate lyase from Fusarium moniliforme: similar modes of chemical and thermal denaturation

A low molecular mass pectate lyase from Fusarium moniliforme was unfolded reversibly by urea and Gdn-HCl at its optimum pH of 8.5, as monitored by intrinsic fluorescence, circular dichroism, and enzymatic activity measurements. Equilibrium unfolding studies yielded a deltaG(H(2)O) of 1.741 kcal/mol, D1/2 of 2.3M, and m value of 0.755kcal/molM with urea and a deltaG(H(2)O) of 1.927kcal/mol, D1/2 of 1.52M, and m value of 1.27 kcal/molM with Gdn-HCl as the denaturant. Thermal denaturation of the pectate lyase at, pH 8.5, was also reversible even after exposure to 75 degrees C for 10 min. Thermodynamic parameters calculated from thermal denaturation curves at pH values from 5.0 to 8.5 yielded a deltaCp of 0.864kcal/(molK). The deltaG(25 degrees C) at, pH 8.5, was 2.06kcal/mol and was in good agreement with the deltaG(H(2)O) values obtained from chemical denaturation curves. There was no exposure of hydrophobic pockets during chemical or thermal denaturation as indicated by the inability of ANS to bind the pectate lyase.     

Fluorescence studies on denaturation and stability of recombinant human interferon-gamma

Unfolding/folding transitions of recombinant human interferon-gamma (hIFNgamma) in urea and guanidine chloride (Gn.HCl) solutions were studied by fluorescence spectroscopy. At pH 7.4 Gn.HCl was a much more efficient denaturant (midpoint of unfolding C* = 1.1 M and deltaG0 = 13.4 kJ/mol) than urea (C* = 2.8 M and deltaG0 = 11.7 kJ/mol). The close deltaG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNgamma is insignificant. Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNgamma remains native in the pH range of 4.8-9.5. Using two quenchers, iodide and acrylamide, and applying the Stern-Volmer equation, a cluster of acidic groups situated in close proximity to the single tryptophan residue was identified. Based on the denaturation experiments at different pH values and on our earlier calculations of the electrostatic interactions in hIFNgamma, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNgamma.     

Analysis of folding and unfolding reactions of cytochrome b5

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

Equilibrium constants under physiological conditions for the reactions of choline kinase and the hydrolysis of phosphorylcholine to choline and inorganic phosphate.

The observed equilibrium constants (Kobs) of the P-choline hydrolysis reaction have been determined under physiological conditions of temperature (38 degrees) and ionic strength (0.25 M) and physiological ranges of pH and free [Mg2+]. Using sigma and square brackets to indicate total concentrations: (see article.) The value of Kobs has been found to be relatively insensitive to variations in pH and free [Mg2+]. At pH 7.0 and taking the standard state of liquid water to have unit activity ([H2O] = 1), Kobs = 26.6 M at free [Mg2+] = 0 [epsilon G0obs = -2.03 kcal/mol(-8.48 kJ/mol)], 26.8 M at free [Mg2+] = 10(-3) M, and 28.4 M at free [Mg2+] = 10(-2) M. At pH 8.0, Kobs = 18.8 M at free [Mg2+] = 0, 19.2 M at free [Mg2+] = 10(-3), and 22.2 M at free [Mg2+] = 10(-2) M. These values apply only to situations where choline and Pi concentrations are both relatively low (such as the conditions found in most tissues). At higher concentrations of phosphate and choline, the value of Kobs becomes significantly increased since HPO42- complexes choline weakly (association constant = 3.3 M-1). The value of K at 38 degrees and I = 0.25 M is calculated to be 16.4 +/- 0.3 M [epsilonG0 = 1.73 kcal/mol (-7.23 kJ/mol)]. The K for the P-choline hydrolysis reaction has been combined with the K for the ATP hydrolysis reaction determined previously under physiological conditions to calculate a value of 4.95 X 10(-3 M [deltaG0 j.28 kcal/mol (13.7 kJ/mol] for the K of the choline kinase reaction (EC 2.7.1.32), an important step in phospholipid metabolism: (see article.) Likewise, values for Kobs for the choline kinase reaction at 38 degrees, pH 7.0, and I = 0.25 M have been calculated to be 5.76 X 10(4) [deltaG0OBS = -6.77 KCAL/MOL (-28.3 KJ/mol)] at [Mg2+] = 0; 1.24 X 10(4) [deltaG0obs = -5.82 kcal/mol (-24.4 kJ/mol)] at [Mg2+] = 10(-3) M and 8.05 X 10(3) [delta G0obs = -5.56 kcal/mol (-23.3 kJ/mol)] at [Mg2+ = 10(-2) M. Attempts to determine the Kobs of the choline kinase reaction directly were unsuccessful because of the high value of the constant. The results indicate that in contrast to the high deltaG0obs for the hydrolysis of the ester bond of acetylcholine, the deltaG0obs for the hydrolysis of the ester bond of P-choline is quite low, among the lowest known for phosphate ester bonds of biological interest.     

Folding of the SARS coronavirus spike glycoprotein immunological fragment (SARS_S1b): thermodynamic and kinetic investigation correlating with three-dimensional structural modeling

Spike glycoprotein of SARS coronavirus (S protein) plays a pivotal role in SARS coronavirus (SARS_CoV) infection. The immunological fragment of the S protein (Ala251-His641, SARS_S1b) is believed to be essential for SARS_CoV entering the host cell through S protein-ACE-2 interaction. We have quantitatively characterized the thermally induced and GuHCl-induced unfolding features of SARS_S1b using circular dichroism (CD), tryptophan fluorescence, and stopped-flow spectral techniques. For the thermally induced unfolding at pH 7.4, the apparent activation energy (E(app)) and transition midpoint temperature (Tm) were determined to be 16.3 +/- 0.2 kcal/mol and 52.5 +/- 0.4 degrees C, respectively. The CD spectra are not dependent on temperature, suggesting that the secondary structure of SARS_S1b has a relatively high thermal stability. GuHCl strongly affected SARS_S1b structure. Both the CD and fluorescent spectra resulted in consistent values of the transition middle concentration of the denaturant (Cm, ranging from 2.30 to 2.45 M) and the standard free energy change (deltaG(o), ranging from 2.1 to 2.5 kcal/mol) for the SARS_S1b unfolding reaction. Moreover, the kinetic features of the chemical unfolding and refolding of SARS_S1b were also characterized using a stopped-flow CD spectral technique. The obvious unfolding reaction rates and relaxation times were determined at various GuHCl concentrations, and the Cm value was obtained, which is very close to the data that resulted from CD and fluorescent spectral determinations. Secondary and three-dimensional structural predictions by homology modeling indicated that SARS_S1b folded as a globular-like structure by beta-sheets and loops; two of the four tryptophans are located on the protein surface, which is in agreement with the tryptophan fluorescence result. The three-dimensional model was also used to explain the recently published experimental results of S1-ACE-2 binding and immunizations.     

Effects of site-specific substitution of 5-fluorouridine on the stabilities of duplex DNA and RNA.

The effects of 5-fluorouridine (FUrd) and 5-fluorodeoxyuridine (FdUrd) substitution on the stabilities of duplex RNA and DNA have been studied to determine how FUrd substitution in nucleic acids may alter the efficiency of biochemical processes that require complementary base pairing for molecular recognition. The parent sequence, 5'-GCGAAUUCGC, contains two non-equivalent uridines. Eight oligonucleotides (four RNA and four DNA) were prepared with either zero, one or two Urd substituted by FUrd. The stability of each self-complementary duplex was determined by measuring the absorbance at 260 nm as a function of temperature. Tm values were calculated from the first derivative of the absorbance versus temperature profiles and values for delta H0 and delta S0 were calculated from the concentration dependence of the Tm. Individual absorbance versus temperature curves were also analyzed by a parametric approach to calculate thermodynamic parameters for the duplex to single-stranded transition. Analysis of the thermodynamic parameters for each oligonucleotide revealed that FUrd substitution had sequence-dependent effects in both A-form RNA and B-form DNA duplexes. Conservation of helix geometry in FUrd-substituted duplexes was determined by CD spectroscopy. FUrd substitution at a single site in RNA stabilized the duplex (delta delta G37 = 0.8 kcal/mol), largely due to more favorable stacking interactions. FdUrd substitution at a single site in DNA destabilized the duplex (delta delta G37 = 0.3 kcal/mol) as a consequence of less favorable stacking interactions. All duplexes melt via single cooperative transitions.     

Calorimetric studies on viroids.

Thermodynamic studies on highly purified viroid preparations were carried out with the help of a very sensitive adiabatic microcalorimeter. Parallel to the change of UV-absorption at 260 nm as a function of temperature, the additional heat capacity of the dilute viroid solution rises sharply within the melting interval, reaches a maximum at T = Tm and declines to a baseline again when the temperature is increased further. From the peak area the molar transition enthalpy can be calculated. The transition enthalpies of citrus exocortis viroid and cucumber pale fruit viroid are 4200 kJ/mol and 3930 kJ/mol, respectively. The calorimetric results are compared to the results obtained from melting studies using UV-absorption.     

Differential scanning calorimetry of bovine rhodopsin in rod-outer-segment disk membranes.

Rhodopsin-containing retinal rod disk membranes from cattle have been examined by differential scanning calorimetry. Under conditions of 67 mM phosphate pH 7.0, unbleached rod outer segment disk membranes gave a single major endotherm with a temperature of denaturation (Tm) of 71.9 +/- 0.4 degrees C and a thermal unfolding calorimetric enthalpy change (delta Hcal) of 700 +/- 17 kJ/mol rhodopsin. Bleached rod outer segment disk membranes (membranes that had lost their absorbance at 498 nm after exposure to orange light) gave a single major endotherm with a Tm of 55.9 +/- 0.3 degrees C and a delta Hcal of 520 +/- 17 kJ/mol opsin. Neither bleached nor unbleached rod outer segment disk membranes gave endotherms upon thermal rescans. When thermal stability is examined over the pH range of 4-9, the major endotherms of both bleached and unbleached rod outer segment disk membranes were found to show maximum stability at pH 6.1. The observed delta Hcal values for bleached and unbleached rod outer segment disk membranes exhibit membrane concentration dependences which plateau at protein concentrations beyond 1.5 mg/mL. For partially bleached samples of rod outer segment disk membranes, the calorimetric enthalpy change for opsin appears to be somewhat dependent on the degree of bleaching, indicating intramembrane nearest neighbor interactions which affect the unfolding of opsin. Delta Hcal and Tm are particularly useful for assessing stability and testing for completeness of regeneration of rhodopsin from opsin. Other factors such as sample preparation and the presence of low concentrations of ethanol also affect the delta Hcal values while the Tm values remain fairly constant. This shows that the delta Hcal is a sensitive parameter for monitoring environmental changes of rhodopsin and opsin.     

Blocking nonspecific adsorption of native food-borne microorganisms by immunomagnetic beads with iota-carrageenan

We present herein the partitioning characteristics of anti-Salmonella and anti-Escherichia coli O157 immunomagnetic beads (IMB) with respect to the nonspecific adsorption of several nontarget food-borne organisms with and without an assortment of well-known blocking agents, such as casein, which have been shown to be useful in other immunochemical applications. We found several common food-borne organisms that strongly interacted with both types of IMB, especially with anti-Salmonella form (av DeltaG0=-20 +/- 4 kJ mol(-1)) even in the presence of casein [1% (w/v): DeltaG0=-18 +/- 3 kJ mol(-1); DeltaDeltaG0 approximately -2 kJ mol(-1)]. However, when one of the most problematic organisms (a native K12-like E. coli isolate; DeltaG0=-19 +/- 2 kJ mol(-1)) was tested for nonspecific binding in the presence of iota-carrageenan (0.03-0.05%), there was an average decline of ca. 90% in the equilibrium capture efficiency xi (DeltaG0=-11 +/- 4 kJ mol(-1); DeltaDeltaG0 approximately -8 kJ mol(-1)). Other anionic polysaccharides (0.1% kappa-carrageenan and polygalacturonic acid) had no significant effect (av DeltaG0=-19 +/- 1 kJ mol(-1); DeltaDeltaG0 approximately 0 kJ mol(-1)). Varying iota-carrageenan from 0% to 0.02% resulted in xi significantly diminishing from 0.69 (e.g., 69% of the cells captured; DeltaG0=-19 +/- 3 kJ mol(-1)) to 0.05 (DeltaG0=-11 +/- 2 kJ mol(-1); DeltaDeltaG0 approximately -9 kJ mol(-1)) at about 0.03% iota-carrageenan where xi leveled off. An optimum blocking ability was achieved with 0.04% iota-carrageenan suspended in 100 mM phosphate buffer. We also demonstrated that the utilization of iota-carrageenan as a blocking agent causes no great loss in the IMBs capture efficiency with respect to the capture of its target organisms, various salmonellae.     

Effect of molecular structure and water content on the dielectric relaxation behaviour of amorphous low molecular weight carbohydrates above and below their glass transition

The dielectric relaxation behaviour of several amorphous low molecular weight carbohydrates and their 10% w/w water mixtures has been studied in the supercooled liquid and glassy regions in the frequency range 100 Hz to 100 kHz. The dry carbohydrates show a primary alpha-relaxation (activation energy 250-405 kJ mol(-1)) at temperatures above the calorimetric glass transition temperature, Tg, and, in most cases, a secondary beta-relaxation (activation energy 42-55 kJ mol(-1)) at sub-Tg temperatures. Whilst D-mannose showed a beta-relaxation similar in strength to D-glucose, its deoxy sugar, L-rhamnose showed a relatively weak beta-relaxation. This indicates that the hydroxymethyl group influences relaxation in carbohydrate glasses. Addition of water shifted the alpha-relaxations to lower temperatures and increased the strength of the beta-relaxations. In glucitol this resulted in a merging of the alpha- and beta-relaxations. The beta-relaxation increased in strength and decreased in temperature for the series of water mixtures: D-glucose, maltose, and maltotriose.     

Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large deltaCp's.

Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8-anilino-1-naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274-329 K. At 298 K, values of deltaGdegrees , deltaCp, and Cm were 3.1+/-0.2 kcal mol(-1), 5.9+/-0.8 kcal mol(-1) K(-1) (15.9 cal (mol-residue)(-1) K(-1)), and 0.8 M, respectively, at pH 3.0 and 14.5+/-0.4 kcal mol(-1), 8.3+/-0.7 kcal mol(-1) K(-1) (22.4 kcal (mol-residue)(-1) K(-1)), and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of deltaGdegrees and deltaCp similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of deltaCp per mol-residue for the molten globule is comparable to corresponding values of deltaCp for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of deltaCp per mol-residue for the unfolding of the native state is among the highest currently known for any protein.     

Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants

The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation <--> X (intermediate) conformation and the second denaturation phase, X conformation <--> D (denatured) conformation are reversible. Conformational characterization of the X state by the far-UV CD, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements led us to conclude that the X state is a molten globule state. Analysis of denaturation transition curves for the stability of different states in terms of Gibbs energy change at pH 6.0 and 25 degrees C led us to conclude that the N state is more stable than the X state by 9.55 +/- 0.32 kcal mol(-1), whereas the X state is more stable than the D state by only 1.40 +/- 0.25 kcal mol(-1). We have also studied the effect of temperature on the equilibria, N conformation <--> X conformation and X conformation <--> D conformation in the presence of different denaturant concentrations using two different optical probes, namely, [theta](222) and Delta epsilon(400). These measurements yielded T(m), (midpoint of denaturation) and Delta H(m) (enthalpy change) at T(m) as a function of denaturant concentration. A plot of Delta H(m) versus corresponding T(m) was used to determine the constant-pressure heat capacity change, Delta C(p) (= ( partial differential Delta H(m)/ partial differential T(m))(p)). Values of Delta C(p) for N conformation <--> X conformation and X conformation <--> D conformation is 0.92 +/- 0.02 kcal mol(-1) K(-1) and 0.41 +/- 0.01 kcal mol(-1) K(-1), respectively. These measurements suggested that about 30% of the hydrophobic groups in the molten globule state are not accessible to the water.     

Temperature sensitivity of calcium binding for parvalbumins from Antarctic and temperate zone teleost fishes

Parvalbumin (PV) is a soluble calcium-binding protein that is especially abundant in fast-twitch muscles of fish and other lower vertebrates. Despite its prevalence in ectothermic taxa, few data address the effects of temperature on PV binding function. In this study, calcium dissociation constants (KD) were measured as a function of temperature (0-25 degrees C) for PV from two Antarctic (Gobionotothen gibberifrons and Chaenocephalus aceratus) and two temperate zone fish species (Cyprinus carpio and Micropterus salmoides). Measurements by fluorometric competitive binding assay show that KD values for PVs from the Antarctic species were significantly higher at all assay temperatures and were less sensitive to temperature relative to carp and bass. However, estimates of KD are fundamentally similar for PVs from the Antarctic and temperate zone species when examined at their native physiological temperature. Variation in pH and ionic strength within a physiologically relevant range had only modest effects on KD. Thermodynamics of calcium binding to PV from G. gibberifrons and C. carpio was measured by isothermal microcalorimetry. When measured at 15 degrees C, the Gibbs free energy change (deltaG) was significantly greater for calcium binding to PV from G. gibberifrons than from carp (-43.4+/-1.5 kJ mol(-1) and -46.6+/-3.0 kJ mol(-1), respectively), and the relative contribution of entropy to deltaG for calcium binding to PV from the Antarctic species was about twice that of carp (deltaS=16.0+/-0.8 J degrees C(-1) mol(-1) for G. gibberifrons; deltaS=7.5+/-0.8 J degrees C(-1) mol(-1) for C. carpio).     

The free energy of dissociation of oligomeric structure in phycocyanin is not linear with denaturant

Using SEC HPLC and fluorescence anisotropy, absorption spectra were assigned to the specific oligomeric structures found with phycocyanin. The absorption spectra were used to quantify the population of each oligomeric form of the protein as a function of both urea concentration and temperature. Phycocyanin hexamers dissociate to trimers with equilibrium constants of 10(-6) to 10(-5). Phycocyanin trimers dissociate to monomers with equilibrium constants of 10(-15) to 10(-12). Both dissociation constants increase linearly with increasing urea concentration, and deltaG(o) values calculated from the equilibrium constants fit best with an exponential function. Our findings appear in contrast with the commonly used linear extrapolation model, deltaG(urea)(o) = deltaG(water)(o) + A[denaturant], in which a linear relationship exists between the free energy of protein unfolding or loss of quaternary structure and the denaturant concentration. Our data examines a smaller range of denaturant concentration than generally used, which might partially explain the inconsistency.     

The interaction of 2,6,8-triaminopurine with poly(uridylic acid).

Equilibrium dialysis measurements have shown that poly(uridylic acid) binds 2,6,8-triaminopurine in a strongly cooperative manner to form a stable 2 : 1 complex at pH 7.8, 0.15 M Na+. The thermal dissociation of the complex has been characterized by ultraviolet absorbance versus temperature profiles. From the variation of Tm with the concentration of uncomplexed triaminopurine at this temperature, the partial molar enthalpy and entropy of formation of the complex have been calculated as --87 (+/- 2) kJ/mol of triaminopurine and --236 (+/- 7) J/mol of triaminopurine per K, respectively. In terms of Tm, the complex is approximately 4 degrees C more stable than the corresponding 2 : 1 complex of poly(U) with 2-aminoadenine. This stabilization is attributed to the existence of an additional hydrogen-bonding interaction, in the poly(U)-triaminopurine complex, between the 8-amino group of 2,6,8-triaminopurine and O(2) of the uracil moiety which is base paired with it in Hoogsteen fashion.     

Thermodynamics of unfolding for turkey ovomucoid third domain: thermal and chemical denaturation.

We have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wave-lengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (Tm) occurs at 58.6 +/- 0.4 degrees C and the enthalpy of unfolding at this temperature (delta Hm) is 40.8 +/- 0.3 kcal/mol. When Tm was perturbed by varying pH and denaturant concentration, the resulting plots of delta Hm versus Tm yield a mean value of 590 +/- 120 cal/(mol.K) for the change in heat capacity upon unfolding (delta Cp). A global fit of the same data to an equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 +/- 110 cal/(mol.K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining delta Cp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520-2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (delta Gu) were plotted versus temperature, the resulting curve appeared linear; therefore, delta Cp could not be determined. However, the data for delta Gu versus denaturant concentration are linear over an extraordinarily wide range of concentrations. Moreover, extrapolated values of delta Gu in urea are identical to values measured directly.     

Glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus. Thermal denaturation and activation.

Pyrococcus furiosus is a marine hyperthermophile that grows optimally at 100 degrees C. Glutamate dehydrogenase (GDH) from P. furiosus is a hexamer of identical subunits and has an M(r) = 270,000 +/- 5500 at 25 degrees C. Electron micrographs showed that the subunit arrangement is similar to that of GDH from bovine liver (i.e. 3/2 symmetry in the form of a triangular antiprism). However, GDH from P. furiosus is inactive at temperatures below 40 degrees C and undergoes heat activation above 40 degrees C. Both NAD+ and NADP+ are utilized as cofactors. Apparently the inactive enzyme also binds cofactors, since the enzyme maintains the ability to bind to an affinity column (Cibacron blue F3GA) and is specifically eluted with NADP+. Conformational changes that accompany activation and thermal denaturation were detected by precision differential scanning microcalorimetry. Thermal denaturation starts at 110 degrees C and is completed at 118 degrees C. delta(cal) = 414 Kcal [mol GDH]-1. Tm = 113 degrees C. This increase in heat capacity indicates an extensive irreversible unfolding of the secondary structure as evidenced also by a sharp increase in absorbance at 280 nm and inactivation of the enzyme. The process of heat activation of GDH from 40 to 80 degrees C is accompanied by a much smaller increase in absorbance at 280 nm and a reversible increase in heat capacity with delta(cal) = 187 Kcal [mol GDH]-1 and Tm = 57 degrees C. This absorbance change as well as the moderate increase in heat capacity suggest that thermal activation leads to some exposure of hydrophobic groups to solvent water as the GDH structure is opened slightly. The increase in absorbance at 280 nm during activation is only 12% of that for denaturation. Overall, GDH appears to be well adapted to correspond with the growth response of P. furiosus to temperature.