Experiment and prediction: a productive symbiosis in studies on the thermodynamics of DNA oligomers.

Recently, we reported the kinetics of hybridization of cDNA dodecamers (Carrillo-Nava, E., Mejía-Radillo, Y., and Hinz, H.-J. Biochemistry 2008, 47, 13153-13157). In this study, we provide the thermodynamic reaction parameters of those dodecamers as well as a comparison with parameters for 24-mers designed from two identical dodecamers in tandem arrangement. The thermodynamic properties were determined by isothermal titration calorimetry (ITC), differential scanning microcalorimetry (DSC), and UV melting studies. On the basis of the results from our kinetic studies, fitting algorithms of DSC and UV melting profiles employed the two-state assumption for the duplex to a single strand dissociation reaction. The formation of both 12-mer and 24-mer duplexes is strongly enthalpy driven at all temperatures. At identical temperatures, the hybridization enthalpy of the 24-mer is within error limits twice that of the 12-mer. Duplex formation is always associated with a significant negative heat capacity change, ΔC(p), which, on a mass basis, is comparable to that observed for protein folding. Only a small part of the favorable reaction enthalpy appears as a standard Gibbs free energy change due to large compensating negative entropy changes linked to duplex formation. On the basis of the results of the present studies, it appears to be absolutely essential for a proper analysis of thermodynamic parameters of oligonucleotide hybridization reactions to combine low temperature ITC measurements of binding enthalpies with DSC and UV melting studies to obtain an accurate assessment of standard Gibbs energy changes or, equivalently, hybridization constants over a broad temperature range. The experimental thermodynamic parameters were compared with theoretical estimates based on nearest-neighbor approximations employing temperature-independent enthalpies. Good agreement between experimental and predicted ΔG° values is observed at ambient temperatures (20-30 °C), as long as helix formation is associated with small molar heat capacity changes. If the experimental ΔC(p) values determined by ITC are taken into account, significant deviations occur.

Dodecamer DNA duplex formation is characterized by second-order kinetics, positive activation energies, and a dependence on sequence and Mg2+ ion concentration.

This study provides quantitative information about the kinetics of formation of a complex between DNA oligomers having 12 bp. The DNA dodecamers were designed in such a way as to avoid the formation of hairpins or slipped duplex structures within single strands. The hybridization was carried out employing stopped-flow techniques. The reaction was studied in different buffers (phosphate or cacodylate), in the presence and absence of Mg2+ ions, and at different temperatures. Under all conditions, the reaction followed second-order kinetics. The association rate constants were on the order of 106 M(-1) s(-1) and were found to increase with an increase in temperature. Both the rate constants and the positive activation energies of the two dodecamers, which differ only by the presence of the TAGG tetrad either at the 3'end or at the 5' end, turned out to be significantly different. The presence of Mg2+ ions had a profound influence on the kinetics of association of either compound by substantially decreasing the activation energy of the process. The dependence on sequence of the kinetics of hybridization was manifest in all parameters under all the experimental conditions.

Association temperature governs structure and apparent thermodynamics of DNA-gold nanoparticles.

Apparent thermodynamics of association of DNA-modified gold nanoparticles has been characterized by UV spectroscopy and dynamic light scattering (DLS). Extinction coefficients of unlabelled and DNA-labelled gold nanoparticles have been determined to permit quantitative analysis of the absorption measurements. In contrast to previous studies the associating gold nanoparticles were furnished with complementary oligonucleotide DNA single strands. This resulted in direct complex formation between the nanoparticles on mixing without the requirement of a DNA linker sequence for initiation of cluster formation. Melting curves of the nanoparticle assemblies formed at different temperatures were subjected to two-state analysis. A comparison of the apparent thermodynamic parameters obtained for the dissociation of these aggregates suggests that both thermodynamically and structurally different nanoparticle clusters are obtained depending on the temperature at which assembly proceeds. The van't Hoff enthalpies permit an estimate of the DNA duplexes: gold nanoparticle ratio involved in network formation.

Application of pressure-modulated differential scanning calorimetry to the determination of relaxation kinetics of multilamellar lipid vesicles.

We report an extension of the recently published PMDSC method that permitted synchronous determination of heat capacity and expansibility when using slow, defined pressure formats in a DSC scan. Here we applied continuously opposing pressure changes that are fast compared to the time constants of the DSC instrument to study relaxation kinetics of phospholipids. Investigations of multilamellar vesicles of DPPC or DSPC in water revealed for both lipids relaxation times of about 30 s at the maximum of the main transition peak and about 15 s at the maximum of the pretransition. The relaxation times in the transition range are proportional to heat capacity of main- and pretransition. The molecular origin of the relaxation processes appears to stem from pressure-induced water fluxes between the interbilayer region and the bulk water phase.

N-acetylanthranilate amidase from Arthrobacter nitroguajacolicus Rü61a, an alpha/beta-hydrolase-fold protein active towards aryl-acylamides and -esters, and properties of its cysteine-deficient variant.

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the alpha/beta-hydrolase-fold superfamily of enzymes; inactivation of (His(6)-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k(cat)/K(m) = 208 mM(-1) s(-1)), while among the aryl-acetylamides, o-carboxy- or o-nitro-substituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His(6)Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His(6)AmqC22A/C63A markedly differed from those of His(6)Amq. The replacements effected some changes in K(m)s of the enzyme and increased k(cat)s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k(cat) for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in alpha-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.

Ca-binding to Bacillus licheniformis alpha-amylase (BLA).

Ca-induced renaturation of Bacillus licheniformis alpha-amylase in the presence of urea has been employed to determine the binding constants of the ion. The native enzyme is folded at 3M urea while the Ca-depleted protein is largely unfolded at this denaturant concentration. Refolding of the protein has been monitored by circular dichroism and the titration curves have been analyzed assuming a model of three independent binding sites. The stoichiometry has been taken from X-ray studies. The refolded protein exhibits a secondary structure that is similar but not identical to that of the native protein. The binding constants have been used to construct a phase diagram that illustrates the contribution of Ca-binding to the resistance against urea unfolding.

Pressure-modulated differential scanning calorimetry: theoretical background.

We demonstrate in this work and in the accompanying paper that it is possible to measure simultaneously heat capacity and expansibility of biomolecules in a single DSC experiment. In this study, we provide the theoretical basis for this new method based on rigorous statistical thermodynamics. The theoretical treatment presented here demonstrates that there are two additive contributions to the heat capacity at variable pressure, viz. (1) the isobaric heat capacity and (2) an expansibility term, and that these contributions can be experimentally separated to obtain simultaneously both heat capacity and expansibility in continuous DSC temperature scans preformed under pressure modulation. Equations that describe the mixed heat capacity and expansibility signal are derived, and experimental strategies as well as data extraction procedures are discussed.

Aggregation and fluorescence quenching of chlorophyll a of the light-harvesting complex II from spinach in vitro.

The salt-induced aggregation of the light-harvesting complex (LHC) II isolated from spinach and its correlation with fluorescence quenching of chlorophyll a is reported. Two transitions with distinctly different properties were observed. One transition related to salt-induced fluorescence quenching takes place at low salt concentration and is dependent both on temperature and detergent concentration. This transition seems to be related to a change in the lateral microorganization of LHCII. The second transition occurs at higher salt concentration and involves aggregation. It is independent of temperature and of detergent at sub-cmc concentrations. During the latter transition the small LHCII sheets (approximately 100 nm in diameter) are stacked to form larger aggregates of approximately 3 microm diameter. Based on the comparison between the physical properties of the transition and theoretical models, direct and specific binding of cations can practically be ruled out as driving force for the aggregation. It seems that in vitro aggregation of LHCII is caused by a complex mixture of different effects such as dielectric and electrostatic properties of the solution and surface charges.

Phase diagrams: a graphical representation of linkage relations.

It is shown here that phase diagrams of ligand-binding biological macromolecules provide a powerful tool for the analysis of reaction mechanisms. The present study provides simple rules for the construction and interpretation of such phase diagrams. We give examples for the derivation of reaction schemes for macromolecules that can bind two different kinds of ligands. By sampling one dimension of a phase diagram it is possible to reconstruct the second dimension, including the correct stoichiometry, positive and negative linkage between the ligands and equilibrium binding constants for the complete series of reactions. The discussion is generalised to temperature and pressure-dependent phase diagrams. To exemplify the new diagram method we analyse the pH-dependent binding of trans-beta-indole acrylic acid to apo-Trp repressor, the pH-dependent thermal denaturation of alpha-chymotrypsinogen A, calcium binding and denaturation of annexin I, high affinity zinc binding to a metallo-beta-lactamase and high-pressure and temperature denaturation of RNase A and staphylococcal nuclease.

Group additivity schemes for the calculation of the partial molar heat capacities and volumes of unfolded proteins in aqueous solution.

A critical review is given of the present state of group additivity schemes for the calculation of partial molar volumes and heat capacities of unfolded proteins. The comparison between the experimental values and the predictions based on the different models shows clearly that only the peptide-based additivity scheme represents properly both the absolute values and the temperature dependence of these thermodynamic quantities.

Activation mechanism of pro-astacin: role of the pro-peptide, tryptic and autoproteolytic cleavage and importance of precise amino-terminal processing.

Astacin (EC 3.4.24.21) is a prototype for the astacin family and for the metzincin superfamily of zinc peptidases, which comprise membrane-bound and secreted enzymes involved in extracellular proteolysis during tissue development and remodelling. Generally, metzincins are translated as pro-enzymes (zymogens), which are activated by removal of an N-terminal pro-peptide. In astacin, however, the mode of zymogen activation has been obscured, since the pro-form does not accumulate in vivo. Here we report the detection of pro-astacin in midgut glands of brefeldin A-treated crayfish (Astacus astacus) by immunoprecipitation and mass spectrometry. We demonstrate that the pro-peptide is able to shield the active site of mature astacin as a transient inhibitor, which is degraded slowly. In vitro studies with recombinant pro-astacin in the absence of another protease reveal a potential of auto-proteolytic activation. The initial cleavage in this autoactivation appears to be an intramolecular event. This is supported by the fact that the mutant E93A-pro-astacin is incapable of autoactivation, and completely resistant to cleavage by mature astacin. However, this mutant is cleaved by Astacus trypsin within the pro-peptide. This probably reflects the in vivo situation, where Astacus trypsin and astacin work together during pro-astacin activation. In a first step, trypsin produces amino-terminally truncated pro-astacin derivatives. These are trimmed subsequently by each other and by astacin to yield the mature amino terminus, which forms a salt-bridge with Glu103 in the active site. The disruption of this salt-bridge in the mutants E103A and E103Q results in extremely heat labile proteins, whose catalytic activities are not altered drastically, however. This supports a concept according to which the linkage of Glu103 to the precisely trimmed amino terminus is a crucial structural prerequisite throughout the astacin family.

Biochemical characterization of the Pseudomonas putida 3-hydroxyacyl ACP:CoA transacylase, which diverts intermediates of fatty acid de novo biosynthesis.

The 3-hydroxyacyl ACP:CoA transacylase (PhaG) was recently identified in various Pseudomonas species and catalyzes the diversion of ACP thioester intermediates of fatty acid de novo biosynthesis toward the respective CoA thioesters, which serve as precursors for polyester and rhamnolipid biosynthesis. PhaG from Pseudomonas putida was overproduced in Escherichia coli as a C-terminal hexahistidine-tagged (His(6)) fusion protein in high yield. The His(6)-PhaG was purified to homogeneity by refolding of PhaG obtained from inclusion bodies, and a new enzyme assay was established. Kinetic analysis of the 3-hydroxyacyl transfer to ACP, catalyzed by His(6)-PhaG, gave K(0.5) values of 28 microm (ACP) and 65 microm (3-hydroxyacyl-CoA) considering V(max) values of 11.7 milliunits/mg and 12.4 milliunits/mg, respectively. A Hill coefficient of 1.38 (ACP) and 1.32 (3-hydroxyacyl-CoA) indicated a positive substrate cooperativity. Subcellular localization studies showed that PhaG is not attached to polyester granules and resides in the cytosol. Gel filtration chromatography analysis in combination with light scattering analysis indicated substrate-induced dimerization of the transacylase. A threading model of PhaG was developed based on the homology to an epoxide hydrolase (1cqz). In addition, the alignment with the alpha/beta-hydrolase fold region indicated that PhaG belongs to alpha/beta-hydrolase superfamily. Accordingly, CD analysis suggested a secondary structure composition of 29% alpha-helix, 22% beta-sheet, 18% beta-turn, and 31% random coil. Site-specific mutagenesis of seven highly conserved amino acid residues (Asp-60, Ser-102, His-177, Asp-182, His-192, Asp-223, His-251) was used to validate the protein model and to investigate organization of the transacylase active site. Only the D182(A/E) mutation was permissive with about 30% specific activity of the wild type enzyme. Furthermore, this mutation caused a change in substrate specificity, indicating a functional role in substrate binding. The serine-specific agent phenylmethylsulfonyl fluoride (PMSF) or the histidine-specific agent diethylpyrocarbonate (DEPC) caused inhibition of 3-hydroxyacyl transfer to holo-ACP, and the S102(A/T) or H251(A/R) PhaG mutant was incapable of catalyzing 3-hydroxyacyl transfer, suggesting that these residues are part of a catalytic triad.

The heat capacity paradox of ligand binding proteins: reconciling the microscopic and macroscopic world.

Differential scanning microcalorimetry (DSC) is a superb method for the analysis of protein energetics. However, the relative simplicity of application has led astray many to assume that a proper analysis of the data was possible without a sound knowledge of the underlying statistical thermodynamic principles. In this study, the question is addressed of how to calculate properly the heat capacity signal of a protein in the presence of high affinity ligands. It is shown that the signal corresponds neither to grand canonic nor to canonic heat capacity. Statistical thermodynamic model calculations result only in the observed macroscopic heat capacity signal, if the protein in the calorimetric cell is assumed to form a grand canonic ensemble (T, p, mu controlled) which is, however, heated under constraints typical for a canonic ensemble (T, p, N controlled). As a consequence, the microscopic statistical thermodynamic heat capacity must be carefully distinguished from the macroscopically observable thermodynamic heat capacity in those cases where proteins unfold in the presence of high affinity ligands.