Biostructural chemistry of magnesium ion: characterization of the weak binding sites on tRNA(Phe)(yeast). Implications for conformational change and activity

The thermodynamics and kinetics of magnesium binding to tRNA(Phe)(yeast) have been studied directly by 25Mg NMR. In 0.17 M Na+(aq), tRNA(Phe) exists in its native conformation and the number of strong binding sites (Ka greater than or equal to 10(4)) was estimated to be 3-4 by titration experiments, in agreement with X-ray structural data for crystalline tRNA(Phe) (Jack et al., 1977). The set of weakly bound ions were in slow exchange and 25Mg NMR resonances were in the near-extreme-narrowing limit. The line shapes of the exchange-broadened magnesium resonance were indistinguishable from Lorentzian form. The number of weak magnesium binding sites was determined to be 50 +/- 8 in the native conformation and a total line-shape analysis of the exchange-broadened 25 Mg2+ NMR resonance gave an association constant Ka of (2.2 +/- 0.2) X 10(2) M-1, a quadrupolar coupling constant (chi B) of 0.84 MHz, an activation free energy (delta G*) of 12.8 +/- 0.2 kcal mol-1, and an off-rate (koff) of (2.5 +/- 0.4) X 10(3) s-1. In the absence of background Na+(aq), up to 12 +/- 2 magnesium ions bind cooperatively, and 73 +/- 10 additional weak binding sites were determined. The binding parameters in the nonnative conformation were Ka = (2.5 +/- 0.2) X 10(2) M-1, chi B = 0.64 MHz, delta G* = 13.1 +/- 0.2 kcal mol-1, and koff = (1.6 +/- 0.4) X 10(3) s-1. In comparison to Mg2+ binding to proteins (chi B typically ca. 1.1-1.6 MHz) the lower chi B values suggest a higher degree of symmetry for the ligand environment of Mg2+ bound to tRNA. A small number of specific weakly bound Mg2+ appear to be important for the change from a nonnative to a native conformation. Implications for interactions with the ribosome 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.     

Differential scanning calorimetric study of the thermal unfolding of Taka-amylase A from Aspergillus oryzae

The thermally induced unfolding of Taka-amylase A, isolated from Aspergillus oryzae, was studied by differential scanning calorimetry. The experimental curves of excess apparent specific heat vs. temperature showed a single asymmetric peak. Curve resolution indicated that this asymmetry is due to the two-state unfolding of three domains in the molecule, with dissociation of the single tightly bound Ca2+ ion occurring during the unfolding of the last domain. Further indication of the dissociation of the specifically bound Ca2+ during denaturation is afforded by the fact that the temperature of maximal excess specific heat, tm, increases with increasing protein concentration in the absence of added excess Ca2+ and with increasing Ca2+ concentration in the presence of added Ca2+. Experiments in a variety of buffers with different enthalpies of ionization indicated that 11.8 +/- 1.5 protons are lost from the protein during unfolding at pH 7.0. In apparent contradiction of this result, the value of tm was found to be essentially independent of pH in the range pH 7-8. No explanation of this anomaly is available. The enthalpy of unfolding at pH 7 and 62 degrees C in the absence of added Ca2+, corrected for the change in buffer protonation, is 2250 +/- 40 kJ mol-1 (42.5 J g-1), and the permanent change in apparent heat capacity is 36.4 +/- 4.1 kJ K-1 mol-1 (0.687 J g-1). Both of these quantities are unusually large for a globular protein.     

Calorimetry of apolipoprotein-A1 binding to phosphatidylcholine-triolein-cholesterol emulsions.

The thermotropic properties of triolein-rich, low-cholesterol dipalmitoyl phosphatidylcholine (DPPC) emulsion particles with well-defined chemical compositions (approximately 88% triolein, 1% cholesterol, 11% diacyl phosphatidylcholine) and particle size distributions (mean diameter, approximately 1000-1100 A) were studied in the absence and presence of apolipoprotein-A1 by a combination of differential scanning and titration calorimetry. The results are compared to egg yolk PC emulsions of similar composition and size. Isothermal titration calorimetry at 30 degrees C was used to saturate the emulsion surface with apo-A1 and rapidly quantitate the binding constants (affinity Ka = 11.1 +/- 3.5 x 10(6) M-1 and capacity N = 1.0 +/- 0.09 apo-A1 per 1000 DPPC) and heats of binding (enthalpy H = -940 +/- 35 kcal mol-1 apo-A1 or -0.92 +/- 0.12 kcal mol-1 DPPC). The entropy of association is -3070 cal deg-1 mol-1 protein or -3 cal deg-1 mol-1 DPPC. Without protein on the surface, the differential scanning calorimetry heating curve of the emulsion showed three endothermic transitions at 24.3 degrees C, 33.0 degrees C, and 40.0 degrees C with a combined enthalpy of 1.53 +/- 0.2 kcal mol-1 DPPC. With apo-A1 on the surface, the heating curve showed the three transitions more clearly, in particular, the second transition became more prominent by significant increases in both the calorimetric and Van't Hoff enthalpies. The combined enthalpy was 2.70 +/- 0.12 kcal mol-1 DPPC and remained constant upon repeated heating and cooling. Indicating that the newly formed DPPC emulsion-Apo-A1 complex is thermally reversible during calorimetry. Thus there is an increase in delta H of 1.17 kcal mol-1 DPPC after apo-A1 is bound, which is roughly balanced by the heat released during binding (-0.92 kcal) of apo-A1. The melting entropy increase, +3.8 cal deg-1 mol-1 DPPC of the three transitions after apo-A1 binds, also roughly balances the entropy (-3 cal deg-1 mol-1 DPPC) of association of apo-A1. These changes indicate that apo-A1 increases the amount of ordered gel-like phase on the surface of DPPC emulsions when added at 30 degrees C. From the stoichiometry of the emulsions we calculate that the mean area of DPPC at the triolein/DPPC interface is 54.5 A2 at 41 degrees C and 54.2 A2 at 30 degrees C. The binding of apo-A1 at 30 degrees C to the emulsion reduces the surface area per DPPC molecule from 54.2 A2 to 50.8 A2. At 30 degrees apo-A1 binds with high affinity and low capacity to the surface of DPPC emulsions and increases the packing density of the lipid domain to which it binds. Apo-A1 was also titrated onto DPPC emulsions at 45 degrees C. This temperature is above the gel liquid crystal transition. No heat was released or adsorbed. Furthermore, egg yolk phosphatidylcholine emulsions of nearly identical composition were also titrated at 30 degrees C with apo-A1 and were euthermic. Association constants were previously measured using a classical centrifugation assay and were used to calculate the entropy of apo-A1 binding (+28 cal deg-1 mol-1 apo-A1). This value indicates that apo-A1 binding to a fluid surface like egg yolk phosphatidylcholine or probably DPPC at 45 degrees C is hydrophobic and is consistent with hydrocarbon lipid or protein moities coming together and excluding water. Thus the binding of apo-A1 to partly crystalline surfaces is entropically negative and increases the order of the already partly ordered phases, whereas binding to liquid surfaces is mainly an entropically driven hydrophobic process.     

The presence of cooperative structures in tryptic fragments of troponin C

Scanning microcalorimetry has been used to study the ability of TR-1 and TR-2 tryptic fragments of troponin C to form an ordered compact structure in solution under different conditions. It has been shown that: (1) in the presence, as well as in the absence of bivalent ions both fragments have a structure which can melt with an intensive heat absorption at heating; (2) the structure of fragment TR-1 containing two Ca2+-specific domains (domains I and II) melts as a whole under all conditions studied and therefore the domains form one cooperative block. Binding of Ca2+ or Mg2+ ions stabilizes the block structure, however, significant conformational rearrangements which would lead to a change of denaturational enthalpy do not occur; (3) Ca2+Mg2+-domains of fragment TR-2 (domains III and IV) represent individual cooperative units, blocks. Stability of these cooperative blocks strongly depends on concentration of bivalent ions and in the presence of 2 mM EDTA the melting temperature of one of them is below 10 degrees. Thermodynamic melting temperature of one of them is below 10 degrees. Thermodynamic melting parameters of cooperative blocks within peptides and in the intact molecule of troponic C are compared.     

Thermodynamic investigations of proteins. IV. Calcium binding protein parvalbumin.

The conformational transitions of calcium binding protein parvalbumin III from carp muscle were studied by scanning calorimetry, potentiometric titration and isothermal calorimetric titration. Changes of Gibbs energy, enthalpy and partial heat capacity were determined. The removal of calcium ions by EDTA is accompanied by 1) a heat absorption of 75 +/- 10 kJ per mole of the protein, 2) a decrease in the Gibbs energy of protein structure stabilisation of about 42 kJ mol-1 and 3) a decrease in thermostability by more than 50 K. The protonation of the acidic groups leads to a loss of calcium followed by denaturation, while the pH of the transition strongly depends on calcium activity. The enthalpy and heat capacity changes at denaturation are comparable with the values observed for other compact globular proteins.     

Mg2+ and Ni2+ ion effect on stability and structure of triple poly I.poly A.poly I helix

The effects of Mg2+ and Ni2+ ions on the absorption spectra of IMP, single-stranded poly I and three-stranded A2I in solutions with 0.1 M Na+ (pH 7) have been studied. In contrast to Mg2+ ions, the Ni2+ ions affect the absorption spectra of these polynucleotides and IMP. The concentration dependences of the intensity at the extrema in the differential UV spectra suggest that in the region of high Ni2+ concentrations ionic complexes with poly I and A2I are formed, which are characterized by the association constants K'I = 2000 M(-1) and K'A2I = 550 M(-1), respectively. The shape of the DUV spectra prompts the conclusion that these complexes are formed due to the inner-sphere interaction of Ni2+ ions with N7 of poly I and A2I presumably due to the outer-sphere Ni2+-O6 interaction. The formation of the complexes leads to destruction of A2I triplexes. The dependences of the melting temperature (T(m)) of A2I on Mg2+ and Ni2+ concentrations have been measured. The thermal stability is observed to increase at the ionic contents up to 0.01 M Mg2+ and only to 2x10(-4) M Ni2+. At higher contents of Ni2+ ions, T(m) lowers and the cooperativity of A2I melting decreases continuously. In all the cases the melting process is the A2I-->A+I+I (3-->1) transition. According to the "ligand" theory, these effects are generated by the energy-advantageous Ni2+ binding to single-stranded poly I (K'A2I < K'I) and by the greater number of binding sites which appears during the 3-->1 transition and is entropy-advantageous.     

Effect of temperature on the creatine kinase equilibrium.

The effect of temperature on the apparent equilibrium constant of creatine kinase (ATP:creatine N-phosphotransferase (EC 2.7.3.2)) was determined. At equilibrium the apparent K' for the biochemical reaction was defined as [formula: see text] The symbol sigma denotes the sum of all the ionic and metal complex species of the reactant components in M. The K' at pH 7.0, 1.0 mM free Mg2+, and ionic strength of 0.25 M at experimental conditions was 177 +/- 7.0, 217 +/- 11, 255 +/- 10, and 307 +/- 13 (n = 8) at 38, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy or heat of the reaction at the specified conditions (delta H' degree) was calculated from a van't Hoff plot of log10K' versus 1/T, and found to be -11.93 kJ mol-1 (-2852 cal mol-1) in the direction of ATP formation. The corresponding standard apparent entropy of the reaction (delta S' degree) was +4.70 J K-1 mol-1. The linear function (r2 = 0.99) between log10 K' and 1/K demonstrates that both delta H' degree and delta S' degree are independent of temperature for the creatine kinase reaction, and that delta Cp' degree, the standard apparent heat capacity of products minus reactants in their standard states, is negligible between 5 and 38 degrees C. We further show from our data that the sign and magnitude of the standard apparent Gibbs energy (delta G' degree) of the creatine kinase reaction was comprised mostly of the enthalpy of the reaction, with 11% coming from the entropy T delta S' degree term. The thermodynamic quantities for the following two reference reactions of creatine kinase were also determined. [formula: see text] The delta H degree for Reaction 2 was -16.73 kJ mol-1 (-3998 cal mol-1) and for Reaction 3 was -23.23 kJ mol-1 (-5552 cal mol-1) over the temperature range 5-38 degrees C. The corresponding delta S degree values for the reactions were +110.43 and +83.49 J K-1 mol-1, respectively. Using the delta H' degree of -11.93 kJ mol-1, and one K' value at one temperature, a second K' at a second temperature can be calculated, thus permitting bioenergetic investigations of organs and tissues using the creatine kinase equilibria over the entire physiological temperature range.     

Determination of monoclonal antibody-induced alterations in Na+/K+-ATPase conformations using fluorescein-labeled enzyme

The fluorescein 5'-isothiocyanate (FITC)-labeled lamb kidney Na+/K+-ATPase has been used to investigate enzyme function and ligand-induced conformational changes. In these studies, we have determined the effects of two monoclonal antibodies, which inhibit Na+/K+-ATPase activity, on the conformational changes undergone by the FITC-labeled enzyme. Monitoring fluorescence intensity changes of FITC-labeled enzyme shows that antibody M10-P5-C11, which inhibits E1 approximately P intermediate formation (Ball, W.J. (1986) Biochemistry 25, 7155-7162), has little effect on the E1 in equilibrium E2 transitions induced by Na+, K+, Mg2+ Pi or Mg2+. ouabain. The M10-P5-C11 epitope, which appears to reside near the ATP-binding site, does not significantly participate in these ligand interactions. In contrast, we find that antibody 9-A5 (Schenk, D.B., Hubert, J.J. and Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951) inhibits both the Na+/K+-ATPase and p-nitrophenylphosphatase activity. Its binding produces a 'Na+-like' enhancement in FITC fluorescence, reduces the ability of K+ to induce the E1 in equilibrium E2 transition and converts E2.K+ to an E1 conformation. Mg2+ binding to the enzyme alters both the conformation of this epitope region and its coupling of ligand interactions. In the presence of Mg2+, 9-A5 binding stabilizes an E1.Mg2+ conformation such that K+-, Pi- and ouabain-induced E1----E2 or E1----E2-Pi transitions are inhibited. Oubain and Pi added together overcome this stabilization. These studies indicate that the 9-A5 epitope participates in the E1 in equilibrium E2 conformational transitions, links Na+-K+ interactions and ouabain extracellular binding site effects to both the phosphorylation site and the FITC-binding region. Antibody-binding studies and direct demonstration of 9-A5 inhibition of enzyme phosphorylation by [32P]Pi confirm the results obtained from the fluorescence studies. Antibody 9-A5 has also proven useful in demonstrating the independence of Mg2+ ATP and Mg2+Pi regulation of ouabain binding. In addition, [3H]ouabain and antibody-binding studies demonstrate that FITC-labeling alters the enzyme's responses to Mg2+ as well as ATP regulation.     

Comparison of the binding of Na+ and Ca2+ to bovine alpha-lactalbumin

alpha-Lactalbumin is a metal-binding protein which binds Ca2+- and Na+-ions competitively to one specific site, giving rise to a large conformational change of the protein. For this reason, the enthalpy change of binding Ca2+ to apo-alpha-lactalbumin (delta Ho) is strongly dependent on the concentration of Na+ ions in the medium. From that relationship a molar enthalpy of -145 +/- 3 kJ X mol-1 is calculated for the Ca2+-binding at pH 7.4 and 25 degrees C, while a delta Ho of -5 +/- 3 kJ X mol-1 is found to substitute a complexed Na+ by a Ca2+-ion. These measurements also allowed us to calculate a binding constant for Na+ of 195 +/- 18 M-1. The molar enthalpy of Na+-loading was found to be -142 +/- 3 kJ X mol-1, a value very close to delta Ho of the binding of Ca2+ to alpha-lactalbumin. Both enthalpy changes in binding Ca2+ and Na+ are independent of the protein concentration. These exothermic values are in agreement with the hypothesis that both Na+- and Ca2+-ions are able to induce the same conformational change in alpha-lactalbumin upon which hydrophobic regions are removed from the solvent, yielding a less hydrophobic protein. The latter is confirmed by means of affinity measurements of the hydrophobic fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulphonate](bis-ANS) to alpha-lactalbumin. The association constant (Ka) decreased from (6.6 +/- 0.5) X 10(4) M-1 in the absence of NaCl to (2.7 +/- 0.2) X 10(4) M-1 in 75 mM NaCl, while the maximum intensity (Imax) of the binary bis-ANS-alpha-lactalbumin complex remained constant at 0.44 +/- 0.02 (arbitrary units). The Ka value of bis-ANS for Ca2+-alpha-lactalbumin was determined at (1.7 +/- 0.2) X 10(4) M-1 and Imax was 0.43 +/- 0.02 (arbitrary units). The difference in hydrophobicity between the two conformational states of the protein was further demonstrated by adsorption experiments of both conformers to phenyl-Sepharose. Apo-alpha-lactalbumin, hydrophobically bound to phenyl-Sepharose, can be eluted by adding Ca2- or Na+-solutions.     

Molar enthalpy change for hydrolysis of phosphorylcreatine under conditions in muscle cells.

The enthalpy change for the hydrolysis of phosphorylcreatine (PCr) by hydrochloric acid or by alkaline phosphatase was observed at 0, 25, and 37 degrees C. The value for delta H is -44 kJ mol-1 under alkaline, Mg2+-free conditions and is almost independent of temperature, ionic strength, and concentration of reactants. In muscle the reaction is accompanied by a transfer of protons from the buffers (largely histidine) to orthophosphate, release of Mg2+ from PCr, and binding of Mg2+ to orthophosphate. Measurements are reported of the heats of these processes. The calculated value of the overall heat of hydrolysis of PCr (including these processes) at pH 7, pMg 3 is -35 kJ mol-1.     

Calcium dependence of Donnan potentials in rigor: the effects of [Mg2+] and anions in isolated rabbit psoas muscle fibres

Contraction in vertebrate striated muscle is known to be dependent upon the binding of calcium ions to the regulatory protein troponin C (TnC). Our electrical (Donnan potential) studies of the subsarcomeric regions have revealed an electrical switching mechanism, which is sensitive to both cation concentration and to particular anions. In a buffer containing phosphate and chloride ions and at 2.7 mM Mg2+ we observe a single charge transition at pCa50 6.8 in both A- and I-bands. At zero Mg2+ the pCa50 of the A-band transition is shifted to 8.0 and the I-band shows two transitions (pCa50 approximately 6.8 and approximately 8.2). Increasing [Mg2+] to 4.5 mM produces a complex effect between pCas 7 and 9 in both bands. All effects are abolished at 9 mM Mg2+. In a chloride-only buffer (imidazole) at zero Mg2+ the direction of the charge transitions is reversed. In addition, two transitions (pCa50 approximately 8.5 and approximately 7.0) are evident in the A-band and three in the I-band (pCa50 approximately 8.5, approximately 7.4, approximately 6.7). In the presence of Mg2+, again the effects of pCa upon the Donnan potential are complex. In the A-band at 2.7 mM Mg2+ two transitions of opposite sign predominate (pCa approximately 7 and approximately 8), whilst in the I band a single transition (pCa approximately 8.3) occurs in the same direction as that observed in phosphate buffer. At 4.5 mM Mg2+ the 'W' shape observed in the corresponding phosphate buffer is preserved in both bands with similar pCa50s. This shape is also apparent in the 9 mM Mg2+ solution. In these two buffer systems, the magnitude of the charge change in terms of electron binding is far larger than expected from simple Ca2+/Mg2+ binding to troponin. In an acetate-only buffer, however, the Donnan potentials of the A-band and I-band were very similar in magnitude and the charge change across the full pCa curve is close to the expected value for Ca2+/Mg2+ binding to troponin. We speculate that titin has a role in the calcium activation of striated muscle in vertebrates for four reasons. First, the effects of long-term storage of the glycerinated muscle; second, the action of [Mg2+]ions; third the effect of anions; and fourth, our published and unpublished observations of sarcomere-length dependence. We also demonstrate the validity of our methodology, relating the charge transitions that we observe to cation-binding studies of a more traditional nature.     

Multistage unfolding of wheat germ ribosomal 5S RNA analyzed by differential scanning calorimetry

Unfolding of purified wheat germ ribosomal RNA has been studied by differential scanning calorimetry (DSC) from 15 to 95 degrees C, in the presence and absence of 100 mM NaCl and/or 10 mM MgCl2. The total enthalpy of melting varies from about 290 (no sodium or magnesium) to 480 kcal/mol (Mg2+ only) and precisely accounts for the number and types of base pairs deduced from prior Fourier-transform infrared experiments. The composite DSC curves are analyzed into four individual two-state melting stages. Both Na+ and Mg2+ shift the melting transitions to higher temperature; in addition, Mg2+ causes the individual transitions to merge (i.e., melt cooperatively) and leads to irreversible chain cleavage at high temperature. The results are analyzed according to three general secondary base-pairing models for eukaryotic 5S RNA.     

Stability, refolding and Ca2+ binding of pullulanase from the hyperthermophilic archaeon Pyrococcus woesei.

The unfolding and refolding of the extremely heat-stable pullulanase from Pyrococcus woesei has been investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 4.86 +/- 0.29 M for intrinsic fluorescence and 4.90 +/- 0.31 M for far-UV CD changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 M guanidinium chloride) was obtained upon removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 M in the first dilution step. Particular attention has been paid to the role of Ca2+ which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca2+ ions with a Kd of 0.080 +/- 0.010 microM and a Hill coefficient H of 1.00 +/- 0.10. This cation enhances significantly the stability of the pullulanase against guanidinium chloride-induced unfolding and the DeltaGH2OD increased from 6.83 +/- 0.43 to 8.42 +/- 0.55 kcal.mol-1. The refolding of the pullulanase, on the other hand, was not affected by Ca2+.     

Domain-domain interactions in high mobility group 1 protein (HMG1).

The high mobility group protein HMG1 is a conserved chromosomal protein with two homologous DNA-binding domains, A and B, and an acidic carboxy-terminal tail, C. The structure of isolated domains A and B has been previously determined by NMR, but the interactions of the different domains within the complete protein were unknown. By means of differential scanning calorimetry and circular dichroism we have investigated the thermal stability of HMG1, of the truncated protein A-B (HMG1 without the acidic tail C) and of the isolated domains A and B. In 3 mm sodium acetate buffer, pH 5, the thermal melting of domains A and B are identical (transition temperature tm = 43 degrees C and 41 degrees C, denaturation enthalpies DeltaH = 46 kcal.mol-1). The thermal melting of protein A-B presents two nearly identical transitions (tm = 40 degrees C and 41 degrees C, DeltaH = 44 kcal.mol-1 and 46 kcal.mol-1, respectively). We conclude that the two domains A and B within protein A-B behave as independent domains. The thermal melting of HMG1 is biphasic. The two transitions have a different value of tm (38 degrees C and 55 degrees C) and corresponding values of DeltaH around 40 kcal.mol-1. We conclude that within HMG1, the acidic tail C is interacting with one of the two domains A and B, however, the two domains A and B do not interact with each other. At 37 degrees C, one of the two domains A and B, within HMG1, is partly unfolded, whereas the other which interacts with the acidic tail C, is fully native. The interaction free energy of the acidic tail C is estimated to be in the range of 2.5 kcal.mol-1 based on simulations of the thermograms of HMG1 as a function of the interaction free energy.     

Thermal stability of PNA/DNA and DNA/DNA duplexes by differential scanning calorimetry

Thermodynamics of the thermal dissociation transitions of 10 bp PNA/DNA duplexes and their corresponding DNA/DNA duplexes in 10 mM sodium phosphate buffer (pH 7.0) were determined from differential scanning calorimetry (DSC) measurements. The PNA/DNA transition temperatures ranged from 329 to 343 K and the calorimetric transition enthalpies ranged from 209 +/- 6 to 283 +/- 37 kJ mol(-1). The corresponding DNA/DNA transition temperatures were 7-20 K lower and the transition enthalpies ranged from 72 +/- 29 to 236 +/- 24 kJ mol(-1). Agreement between the DSC and UV monitored melting (UVM) determined transition enthalpies validated analyzing the UVM transitions in terms of a two-state transition model. The transitions exhibited reversibility and were analyzed in terms of an AB = A + B two-state transition model which yielded van't Hoff enthalpies in agreement with the transition enthalpies. Extrapolation of the transition enthalpies and free energy changes to ambient temperatures yielded more negative values than those determined directly from isothermal titration calorimetry measurements on formation of the duplexes. This discrepancy was attributed to thermodynamic differences in the single-strand structures at ambient and at the transition temperatures, as indicated by UVM measurements on single DNA and PNA strands.     

Entropic drive in the sarcoplasmic reticulum ATPase interaction with Mg2+ and Pi.

Thermodynamic quantities for the binding of Mg2+ (in the presence of Ca2+) and Pi (in the presence of Mg2+ and absence of Ca2+) to sarcoplasmic reticulum ATPase were determined from isothermal titration calorimetry measurements at 25 degrees C. Mg2+ and Pi are involved in reversal of the ATPase hydrolytic reaction, and their interactions with the ATPase are conveniently studied under equilibrium conditions. We found that the Mg2+ binding reaction is endothermic with a binding constant (Kb) = 142 +/- 4 M(-1), a binding enthalpy of 180 +/- 3 kJ mol(-1), and an entropy contribution (TdeltaSb) = 192 +/- 3 kJ mol(-1). Similarly, Pi binding is also an endothermic reaction with Kb = 167 +/- 17 M(-1), deltaHb = 65.3 +/- 5.4 kJ mol(-1), and TdeltaSb = 77.9 +/- 5.4 kJ mol(-1). Our measurements demonstrate that the ATPase can absorb heat from the environment upon ligand binding, and emphasize the important role of entropic mechanisms in energy transduction by this enzyme.     

A comparison of the mechanisms of CO2 hydration by native and Co2+-substituted carbonic anhydrase II

We have measured the pH dependence of kcat and kcat/Km for CO2 hydration catalyzed by both native Zn2+-and metallo-substituted Co2+-bovine carbonic anhydrase II in the absence of inhibitory ions. For the Zn2+-enzyme, the pKa values controlling kcat and kcat/Km profiles are similar, but for the Co2+-enzyme the values are about 0.6 pH units apart. Computer simulations of a metal-hydroxide mechanism of carbonic anhydrase suggest that the data for both native and Co2+-carbonic anhydrase can be accounted for by the same mechanism of action, if we postulate that the substitution of Co2+ for Zn2+ in the active site causes a separation of about 0.6 pH units in the pKa values of His-64 and the metal-bound water molecule. We have also measured the activation parameters for kcat and kcat/Km for Co2+-substituted carbonic anhydrase II-catalyzed CO2 hydration and have compared these values to those obtained previously for the native Zn2+-enzyme. For kcat and kcat/Km we obtain an enthalpy of activation of 4.4 +/- 0.6 and approximately 0 kcal mol-1, respectively. The corresponding entropies of activation are -18 +/- 2 and -27 +/- 2 cal mol-1 K-1.     

Thermodynamics of the hydrolysis of sucrose

A thermodynamic investigation of the hydrolysis of sucrose to fructose and glucose has been performed using microcalorimetry and high-pressure liquid chromatography. The calorimetric measurements were carried out over the temperature range 298-316 K and in sodium acetate buffer (0.1 M, pH 5.65). Enthalpy and heat capacity changes were obtained for the hydrolysis of aqueous sucrose (process A): sucrose(aq) + H2O(liq) = glucose(aq) + fructose (aq). The determination of the equilibrium constant required the use of a thermochemical cycle calculation involving the following processes: (B) glucose 1-phosphate2-(aq) = glucose 6-phosphate2-(aq); (C) sucrose(aq) + HPO4(2-)(aq) = glucose 1-phosphate2-(aq) + fructose(aq); and (D) glucose 6-phosphate2-(aq) + H2O(liq) = glucose(aq) + HPO4(2-)(aq). The equilibrium constants determined at 298.15 K for processes B and C are 17.1 +/- 1.0 and 32.4 +/- 3.0, respectively. Equilibrium data for process D was obtained from the literature, and in conjunction with the data for processes B and C, used to calculate a value of the equilibrium constant for the hydrolysis of aqueous sucrose. Thus, for process A, delta G0 = -26.53 +/- 0.30 kJ mol-1, K0 = (4.44 +/- 0.54) x 10(4), delta H0 = -14.93 +/- 0.16 kJ mol-1, delta So = 38.9 +/- 1.2 J mol-1 K-1, and delta CoP = 57 +/- 14 J mol-1 K-1 at 298.15 K. Additional thermochemical cycles that bear upon the accuracy of these results are examined.