Stability, unfolding mechanism, spectroscopic, densimetric, and structural characteristics of the oxidatively stable C69S variant (HodC) of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) have been determined by classical and pressure modulation scanning calorimetry (DSC and PMDSC, respectively), circular dichroism (CD) spectroscopy, differential scanning densimetry (DSD), and dynamic light scattering measurements. At 25 degrees C, hexahistidine-tagged HodC has a hydrodynamic radius of 2.3 nm and is characterized by an unusually high degree of alpha-helical structure of approximately 60%, based on deconvolution of CD spectra. The percentage of beta-sheets and -turns is expected to be relatively low in view of its sequence similarity to proteins of the alpha/beta-hydrolase fold superfamily. His6HodC exhibits three-state unfolding (N <--> I <--> D) with an intermediate state I that exhibits at the transition temperature a volume larger than that of the native or denatured state. The intermediate state I is also associated with the highest isothermal expansion coefficient, alphaP, of the three states and exhibits a significantly lower percentage of alpha-helical structure than the native state. The stability difference between the native and intermediate state is rather small which makes I a potential candidate for reactions with various ligands, particularly those having a preference for the apparently preserved beta-type motifs.
A new method is described that permits the continuous and synchronous determination of heat capacity and expansibility data. We refer to it as pressure-modulated differential scanning calorimetry (PMDSC), as it involves a standard DSC temperature scan and superimposes on it a pressure modulation of preselected format. The power of the method is demonstrated using salt solutions for which the most accurate heat capacity and expansibility data exist in the literature. As the PMDSC measurements could reproduce the parameters with high accuracy and precision, we applied the method also to an aqueous suspension of multilamellar DSPC vesicles for which no expansibility data had been reported previously for the transition region. Excellent agreement was obtained between data from PMDSC and values from independent direct differential scanning densimetry measurements. The basic theoretical background of the method when using sawtooth-like pressure ramps is given under Supporting Information, and a complete statistical thermodynamic derivation of the general equations is presented in the accompanying paper.
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.
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