Interactions between N- and C-terminal domains of the Saccharomyces cerevisiae high-mobility group protein HMO1 are required for DNA bending

The Saccharomyces cerevisiae high-mobility group protein HMO1 is composed of two DNA-binding domains termed box A and box B, of which only box B is predicted to adopt a HMG fold, and a lysine-rich C-terminal extension. To assess the interaction between individual domains and their contribution to DNA binding, several HMO1 variants were analyzed. Using circular dichroism spectroscopy, thermal stability was measured. While the melting temperatures of HMO1-boxA and HMO1-boxB are 57.2 and 47.2 degrees C, respectively, HMO1-boxBC, containing box B and the entire C-terminal tail, melts at 46.1 degrees C, suggesting little interaction between box B and the tail. In contrast, full-length HMO1 exhibits a single melting transition at 47.9 degrees C, indicating that interaction between box A and either box B or the tail destabilizes this domain. As HMO1-boxAB, lacking only the lysine-rich C-terminal segment, exhibits two melting transitions at 46.0 and 63.3 degrees C, we conclude that the destabilization of the box A domain seen in full-length HMO1 is due primarily to its interaction with the lysine-rich tail. Determination of DNA substrate specificity using electrophoretic mobility shift assays shows unexpectedly that the lysine-rich tail does not increase DNA binding affinity but instead is required for DNA bending by full-length HMO1; HMO1-boxBC, lacking the box A domain, also fails to bend DNA. In contrast, both HMO1 and HMO1-boxAB, but not the individual HMG domains, exhibit preferred binding to constrained DNA minicircles. Taken together, our data suggest that interactions between box A and the C-terminal tail induce a conformation that is required for DNA bending.     

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

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

Nature of full-length HMGB1 binding to cisplatin-modified DNA

HMGB1, a highly conserved non-histone DNA-binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The interaction of full-length HMGB1, containing two tandem HMG box domains and a C-terminal acidic tail, with cisplatin-modified DNA was investigated by hydroxyl radical footprinting and electrophoretic gel mobility shift assays. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A, as revealed by footprinting, with a dissociation constant K(d) of 120 nM. Site-directed mutagenesis of intercalating residues in both HMG domains A and B in full-length HMGB1 further supports the conclusion that only one HMG box domain is bound to the site of cisplatin damage. Interaction of the C-terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. These results illuminate the respective roles of the tandem HMG boxes and the C-terminal acidic tail of HMGB1 in binding to DNA and to the major DNA adducts formed by the anticancer drug cisplatin.     

Thermodynamics of ribonuclease T1 denaturation.

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

Redox properties of mesophilic and hyperthermophilic rubredoxins as a function of pressure and temperature.

The formal equilibrium reduction potentials of recombinant electron transport protein, rubredoxin (MW = 7500 Da), from both the mesophilic Clostridium pasteurianum (Topt = 37 degrees C) and hyperthermophilic Pyrococcus furiosus (Topt = 95 degrees C) were recorded as a function of pressure and temperature. Measurements were made utilizing a specially designed stainless steel electrochemical cell that easily maintains pressures between 1 and 600 atm and a temperature-controlled cell that maintains temperatures between 4 and 100 degrees C. The reduction potential of P. furiosus rubredoxin was determined to be 31 mV at 25 degrees C and 1 atm, -93 mV at 95 degrees C and 1 atm, and 44 mV at 25 degrees C and 400 atm. Thus, the reduction potential of P. furiosus rubredoxin obtained under standard conditions is likely to be dramatically different from the reduction potential obtained under its normal operating conditions. Thermodynamic parameters associated with electron transfer were determined for both rubredoxins (for C. pasteurianum, DeltaV degrees = -27 mL/mol, DeltaS degrees = -36 cal K-1 mol-1, and DeltaH degrees = -10 kcal/mol, and for P. furiosus, DeltaV degrees = -31 mL/mol, DeltaS degrees = -41 cal K-1 mol-1, and DeltaH degrees = -13 kcal/mol) from its pressure- and temperature-reduction potential profiles. The thermodynamic parameters for electron transfer (DeltaV degrees, DeltaS degrees, and DeltaH degrees ) for both proteins were very similar, which is not surprising considering their structural similarities and sequence homology. Despite the fact that these two proteins exhibit dramatic differences in thermostability, it appears that structural changes that confer dramatic differences in thermostability do not significantly alter electron transfer reactivity. The experimental changes in reduction potential as a function of pressure and temperature were simulated using a continuum dielectric electrostatic model (DELPHI). A reasonable estimate of the protein dielectric constant (epsilonprotein) of 6 for both rubredoxins was determined from these simulations. A discussion is presented regarding the analysis of electrostatic interaction energies of biomolecules through pressure- and temperature-controlled electrochemical studies.     

The HMG1 ta(i)le

We have studied structural changes in DNA/protein complexes using the CD spectroscopy, upon the interaction of HMG1-domains with calf thymus DNA at different ionic strengths. HMG1 protein isolated from calf thymus and recombinant HMG1-(A+B) protein were used. Recombinant protein HMG1-(A+B) represents a rat HMG1 lacking C-terminal acidic tail. At low ionic strength (15 mM NaCl) we observed similar behavior of both proteins upon interaction with DNA. Despite this, at higher ionic strength (150 mM NaCl) their interaction with DNA leads to a completely different structure of the complexes. In the case of HMG1-(A+B)/DNA complexes we observed the appearance of DNA fractions possessing very high optical activity. This could be a result of formation of the highly-ordered DNA structures modulated by the interaction with HMG1-domains. Thus the comparison studies of HMG1 and HMG1-(A+B) interaction with DNA show that negatively charged C-terminal tail of HMG1 modulates interaction of the protein with DNA. The striking difference of the behaviour of these two systems allows us to explain the functional role of multiple HMG1 domains in some regulatory and architectural proteins.     

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 long acidic tail of high mobility group box 1 (HMGB1) protein forms an extended and flexible structure that interacts with specific residues within and between the HMG boxes

HMGB1 (high mobility group B1) is a conserved chromosomal protein composed of two similar DNA binding domains (HMG box A and box B) linked by a short basic stretch to an acidic C-terminal tail of 30 residues. The acidic tail modulates the DNA binding properties of HMGB1, and its length differentiates the various HMGB family members. We synthesized a peptide that corresponds to the acidic tail in HMGB1 (T-peptide) and studied its binding to the single boxes and to the fragment corresponding to tailless HMGB1 (designated as AB(bt) fragment). CD spectroscopy showed that T-peptide stabilizes significantly the AB(bt) fragment and that the complex has an identical thermal stability as full-length HMGB1. Calorimetric and NMR data showed that T-peptide binds with a dissociation constant of 9 microM to box A and much more weakly to box B. (1)H-(15)N HSQC spectra of full-length HMGB1 and of the AB(bt) fragment are very similar; the small chemical shift differences that exist correspond to those residues of the AB(bt) fragment that were affected by the addition of the T-peptide. We conclude that the T-peptide mimics closely the acidic tail and that the basic stretch and the acidic tail form an extended and flexible segment. The tail interacts with specific residues in the boxes and shields them from other interactions.     

DNA looping by the HMG-box domains of HMG1 and modulation of DNA binding by the acidic C-terminal domain.

We have compared HMG1 with the product of tryptic removal of its acidic C-terminal domain termed HMG3, which contains two 'HMG-box' DNA-binding domains. (i) HMG3 has a higher affinity for DNA than HMG1. (ii) Both HMG1 and HMG3 supercoil circular DNA in the presence of topoisomerase I. Supercoiling by HMG3 is the same at approximately 50 mM and approximately 150 mM ionic strength, as is its affinity for DNA, whereas supercoiling by HMG1 is less at 150 mM than at 50 mM ionic strength although its affinity for DNA is unchanged, showing that the acidic C-terminal tail represses supercoiling at the higher ionic strength. (iii) Electron microscopy shows that HMG3 at a low protein:DNA input ratio (1:1 w/w; r = 1), and HMG1 at a 6-fold higher ratio, cause looping of relaxed circular DNA at 150 mM ionic strength. Oligomeric protein 'beads' are apparent at the bases of the loops and at cross-overs of DNA duplexes. (iv) HMG3 at high input ratios (r = 6), but not HMG1, causes DNA compaction without distortion of the B-form. The two HMG-box domains of HMG1 are thus capable of manipulating DNA by looping, compaction and changes in topology. The acidic C-tail down-regulates these effects by modulation of the DNA-binding properties.     

Thermal denaturation of T4 gene 32 protein: effects of zinc removal and substitution

Gene 32 protein (g32P), the single-stranded (ss) DNA binding protein from bacteriophage T4, is a zinc metalloprotein. The intrinsic zinc is one of the factors required for the protein to bind cooperatively to a ssDNA lattice. We have used differential scanning calorimetry to determine how the thermodynamic parameters characterizing the denaturation of g32P are affected by removal or substitution of the intrinsic zinc. Over a wide concentration range (1-10 mg/mL), the native Zn(II) protein unfolds at a tm of 55 degrees C with an associated mean enthalpy change of 139 kcal mol-1. Under the same conditions, the metal-free apoprotein denatures over a relatively broader temperature range centered at 49 degrees C, with a mean enthalpy change of 84 kcal mol-1. Substitution of Zn(II) in g32P by either Cd(II) or Co(II) does not significantly change the enthalpy of denaturation but does affect the thermal stability of the protein. All metallo forms of g32P when bound to poly(dT) undergo highly cooperative denaturational transitions characterized by asymmetric differential scanning calorimetry peaks with increases in tm of 4-5 degrees C compared to the unliganded metalloprotein. Removal of the metal ion from g32P significantly reduces the cooperativity of binding to poly(dT) [Giedroc, D. P., Keating, K. M., Williams, K. R., & Coleman, J. E. (1987) Biochemistry 26, 5251-5259], and presumably as a consequence of this, apo-g32P shows no change in either the shape or the midpoint of the thermal transition on binding to poly(dT).(ABSTRACT TRUNCATED AT 250 WORDS)     

A monofunctional and thermostable prephenate dehydratase from the archaeon Methanocaldococcus jannaschii

Prephenate dehydratase (PDT) is an important but poorly characterized enzyme that is involved in the production of L-phenylalanine. Multiple-sequence alignments and a phylogenetic tree suggest that the PDT family has a common structural fold. On the basis of its sequence, the PDT from the extreme thermophile Methanocaldococcus jannaschii (MjPDT) was chosen as a promising representative of this family for pursuing structural and functional studies. The corresponding pheA gene was cloned and expressed in Escherichia coli. It encodes a monofunctional and thermostable enzyme with an N-terminal catalytic domain and a C-terminal regulatory ACT domain. Biophysical characterization suggests a dimeric (62 kDa) protein with mixed alpha/beta secondary structure elements. MjPDT unfolds in a two-state manner (Tm = 94 degrees C), and its free energy of unfolding [DeltaGU(H2O)] is 32.0 kcal/mol. The purified enzyme catalyzes the conversion of prephenate to phenylpyruvate according to Michaelis-Menten kinetics (kcat = 12.3 s-1 and Km = 22 microM at 30 degrees C), and its activity is pH-independent over the range of pH 5-10. It is feedback-inhibited by L-phenylalanine (Ki = 0.5 microM), but not by L-tyrosine or L-tryptophan. Comparison of its activation parameters (DeltaH(++)= 15 kcal/mol and DeltaS(++)= -3 cal mol-1 K-1) with those for the spontaneous reaction (DeltaH(++) = 17 kcal/mol and DeltaS(++)= -28 cal mol-1 K-1) suggests that MjPDT functions largely as an entropy trap. By providing a highly preorganized microenvironment for the dehydration-decarboxylation sequence, the enzyme may avoid the extensive solvent reorganization that accompanies formation of the carbocationic intermediate in the uncatalyzed reaction.     

Conformational stability of HPr: the histidine-containing phosphocarrier protein from Bacillus subtilis.

The conformational stability of the histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been determined using a combination of thermal unfolding and solvent denaturation experiments. The urea-induced denaturation of HPr was monitored spectroscopically at fixed temperatures and thermal unfolding was performed in the presence of fixed concentrations of urea. These data were analyzed in several different ways to afford a measure of the cardinal parameters (delta Hg, Tg, delta Sg, and delta Cp) that describe the thermodynamics of folding for HPr. The method of Pace and Laurents (Pace CN, Laurents DV, 1989, Biochemistry 28:2520-2525) was used to estimate delta Cp as was a global analysis of the thermal- and urea-induced unfolding data. Each method used to analyze the data gives a similar value for delta Cp (1,170 +/- 50 cal mol-1K-1). Despite the high melting temperature for HPr (Tg = 73.5 degrees C), the maximum stability of the protein, which occurs at 26 degrees C, is quite modest (delta Gs = 4.2 kcal mol-1). In the presence of moderate concentrations of urea, HPr exhibits cold denaturation, and thus a complete stability curve for HPr, including a measure of delta Cp, can be achieved using the method of Chen and Schellman (Chen B, Schellman JA, 1989, Biochemistry 28:685-691). A comparison of the different methods for the analysis of solvent denaturation curves is provided and the effects of urea on the thermal stability of this small globular protein are discussed. The methods presented will be of general utility in the characterization of the stability curve for many small proteins.     

Kinetics and thermodynamics of double-helix formation of self-complementary deoxyribooctanucleotides d(TCTATAGA) and d(TAGATCTA).

Double-helix formation of self-complementary deoxyribooctanucleotides, d(TCTATAGA) and d(TAGATCTA), with identical nearest neighbor base pairs has been studied by means of UV melting and temperature-jump techniques. The self-complementary duplexes of both octanucleotides with identical nearest neighbors had similar stabilities: The stabilization energies of the octanucleotides at 25 degrees C were 5.8 kcal mol-1 for d(TCTATAGA) and 6.7 kcal mol-1 for d(TAGATCTA). On the kinetic curve the melting reactions finished within 20 ms for d(TCTATAGA) and 40 ms for d(TAGATCTA) at 20 degrees C. For both octanucleotides the rate constants of dissociation increased and the rate constants of association decreased with increasing temperature.     

Global structural rearrangement of the cell penetrating ribonuclease colicin E3 on interaction with phospholipid membranes

Nuclease type colicins and related bacteriocins possess the unprecedented ability to translocate an enzymatic polypeptide chain across the Gram-negative cell envelope. Here we use the rRNase domain of the cytotoxic ribonuclease colicin E3 to examine the structural changes on its interaction with the membrane. Using phospholipid vesicles as model membranes we show that anionic membranes destabilize the nuclease domain of the rRNase type colicin E3. Intrinsic tryptophan fluorescence and circular dichroism show that vesicles consisting of pure DOPA act as a powerful protein denaturant toward the rRNase domain, although this interaction can be entirely prevented by the addition of salt. Binding of E3 rRNase to DOPA vesicles is an endothermic process (DeltaH=24 kcal mol-1), reflecting unfolding of the protein. Consistent with this, binding of a highly destabilized mutant of the E3 rRNase to DOPA vesicles is exothermic. With mixed vesicles containing anionic and neutral phospholipids at a ratio of 1:3, set to mimic the charge of the Escherichia coli inner membrane, destabilization of E3 rRNase is lessened, although the melting temperature of the protein at pH 7.0 is greatly reduced from 50 degrees C to 30 degrees C. The interaction of E3 rRNase with 1:3 DOPA:DOPC vesicles is also highly dependent on both ionic strength and temperature. We discuss these results in terms of the likely interaction of the E3 rRNase and the related E9 DNase domains with the E. coli inner membrane and their subsequent translocation to the cell cytoplasm.     

Structural requirements for cooperative binding of HMG1 to DNA minicircles

DNA minicircles, where the length of DNA is below the persistence length, are highly effective, preferred, ligands for HMG-box proteins. The proteins bind to them "structure-specifically" with affinities in the nanomolar range, presumably to an exposed widened minor groove. To understand better the basis of this preference, we have studied the binding of HMG1 (which has two tandem HMG boxes linked by a basic extension to a long acidic tail) and Drosophila HMG-D (one HMG box linked by a basic region to a short and less acidic tail), and their HMG-box domains, to 88 bp and 75 bp DNA minicircles. In some cases we see cooperative binding of two molecules to the circles. The requirements for strong cooperativity are two HMG boxes and the basic extension; the latter also appears to stabilize and constrain the complex, preventing binding of further protein molecules. HMG-D, with a single HMG box, does not bind cooperatively. In the case of HMG1, the acidic tail is not required for cooperativity and does not affect binding significantly, in contrast to a much greater effect with linear DNA, or even four-way junctions (another distorted DNA substrate). Such effects could be relevant in the hierarchy of binding of HMG-box proteins to DNA distortions in vivo, where both single-box and two-box proteins might co-exist, with or without basic extensions and acidic tails.     

Association of terminal complement proteins in solution and modulation by suramin.

The association of terminal complement proteins was investigated by analytical ultracentrifugation and multi-angle laser light scattering. Native C8 and C9 formed a heterodimer in solution of physiological ionic strength with a free-energy change DeltaG degrees of -8.3 kcal/mol and a dissociation constant Kd of 0.6 microM (at 20 degrees C) that was ionic strength- and temperature-dependent. A van't Hoff plot of the change in Kd was linear between 10 and 37 degrees C and yielded values of DeltaH degrees = -12.9 kcal/mol and DeltaS degrees = -15.9 cal mol-1 deg-1, suggesting that electrostatic forces play a prominent role in the interaction of C8 with C9. Native C8 also formed a heterodimer with C5, and low concentrations of polyionic ligands such as protamine and suramin inhibited the interaction. Suramin induced high-affinity trimerization of C8 (Kd = 0.10 microM at 20 degrees C) and dimerization of C9 (Kd = 0.86 microM at 20 degrees C). Suramin-induced C8 oligomerization may be the primary reason for the drug's ability to prevent complement-mediated hemolysis. Analysis of sedimentation equilibria and also of the fluorescence enhancement of suramin when bound to protein provided evidence for two suramin-binding sites on each C9 and three on each C8 in the oligomers. Oligomerization could be reversed by high suramin concentrations, but 8-aminonaphthalene-1,3,6- trisulfonate (ANTS2- ), which mimics half a suramin molecule, could not compete with suramin binding and oligomerization suggesting that the drug also binds nonionically to the proteins.     

Thermodynamics of HMGB1 interaction with duplex DNA

The high mobility group protein HMGB1 is a small, highly abundant protein that binds to DNA in a non-sequence-specific manner. HMGB1 consists of 2 DNA binding domains, the HMG boxes A and B, followed by a short basic region and a continuous stretch of 30 glutamate or aspartate residues. Isothermal titration calorimetry was used to characterize the binding of HMGB1 to the double-stranded model DNAs poly(dAdT).(dTdA) and poly(dGdC).(dCdG). To elucidate the contribution of the different structural motifs to DNA binding, calorimetric measurements were performed comparing the single boxes A and B, the two boxes plus or minus the basic sequence stretch (AB(bt) and AB), and the full-length HMGB1 protein. Thermodynamically, binding of HMGB1 and all truncated constructs to duplex DNA was characterized by a positive enthalpy change at 15 degrees C. From the slopes of the temperature dependence of the binding enthalpies, heat capacity changes of -0.129 +/- 0.02 and -0.105 +/- 0.05 kcal mol(-1) K(-1) were determined for box A and full-length HMGB1, respectively. Significant differences in the binding characteristics were observed using full-length HMGB1, suggesting an important role for the acid tail in modulating DNA binding. Moreover, full-length HMGB1 binds differently these two DNA templates: binding to poly(dAdT).(dTdA) was cooperative, had a larger apparent binding site size, and proceeded with a much larger unfavorable binding enthalpy than binding to poly(dGdC).(dCdG).     

Conformational states of the nuclear GTP-binding protein Ran and its complexes with the exchange factor RCC1 and the effector protein RanBP1.

It has been shown before by (31)P NMR that Ras bound to the nonhydrolyzable GTP analogue guanosine 5'-O-(beta, gamma-imidotriphosphate) (GppNHp) exists in two conformations which are rapidly interconverting with a rate constant of 3200 s-1 at 30 degrees C [Geyer, M., et al. (1996) Biochemistry 35, 10308-10320]. Here we show that Ran complexed with GTP also exists in two conformational states, 1 and 2, which can be directly inferred from the occurrence of two (31)P NMR resonance lines for the gamma-phosphate group of bound GTP. The exchange between the two states is slow on the NMR time scale with a value of <200 s-1 at 5 degrees C for the corresponding first-order rate constants. In wild-type Ran, the equilibrium constant K' between the two states is 0.7 at 278 K, is different for various mutants, and is strongly dependent on the temperature. The standard enthalpy DeltaH degrees and the standard entropy DeltaS degrees for the conformational transitions determined from the NMR spectra are as follows: DeltaH degrees = 37 kJ mol-1 and DeltaS degrees = 130 J mol-1 K-1 for wild-type Ran.GTP. In complex with the Ran-binding protein RanBP1, one of the Ran.GTP conformations (state 2) is stabilized. The interaction of Ran with the guanine nucleotide exchange factor protein RCC1 was also studied by (31)P NMR spectroscopy. In the presence of nucleotide, the ternary complex of Ran.nucleotide.RCC1, an intermediate in the guanine nucleotide exchange reaction, could be observed. A model for the conformational transition of Ran.GTP is proposed where the two states observed are caused by the structural flexibility of the effector loop of Ran; in solution, state 2 resembles the GTP-bound form found in the crystal structure of the Ran-RanBP complex.     

Kinetic and thermodynamic investigation on ascorbate oxidase activity and stability of a Cucurbita maxima extract

The kinetic and thermodynamic properties of ascorbate oxidase (AO) activity and stability of a Cucurbita maxima extract were investigated. Activity tests performed at 25 degrees C using initial ascorbic acid concentration in the range 50-750 M allowed estimating the Michaelis constant for this substrate (Km = 126 microM) and the maximum initial rate of ascorbic acid oxidation (A0,max = 1.57 mM min-1). The main thermodynamic parameters of the enzyme reaction (DeltaH* = 10.3 kJ mol-1; DeltaG* = 87.2 kJ mol-1; DeltaS* = -258 J mol-1 K-1) were estimated through activity tests performed at 25-48 C. Within such a temperature range, no decrease in the initial reaction rate was detected. The long-term thermostability of the raw extract was then investigated by means of residual activity tests carried out at 10-70 degrees C, which allowed estimating the thermodynamic parameters of the irreversible enzyme inactivation as well (DeltaH*D = 51.7 kJ mol-1; DeltaG*D = 103 kJ mol-1; S*D = -160 J mol-1 K-1). Taking into account the specific rate of AO inactivation determined at different temperatures, we also estimated the enzyme half-life (1047 min at 10 degrees C and 21.2 min at 70 degrees C) and predicted the integral activity of a continuous system using this enzyme preparation. This work should be considered as a preliminary attempt to characterize the AO activity of a C. maxima extract before its concentration by liquid-liquid extraction techniques.