Stabilization energy of the compact Caf1<Subscript>13–149</Subscript> subunit from <Emphasis Type="Italic">Yersinia pestis</Emphasis>

It is shown by several methods (circular dichroism, viscometry, intrinsic fluorescence, and fluorescence of labels) that, as in the case of small globular proteins, the folding-unfolding transition in the Caf1_13–149 subunit under the action of two denaturants (urea and 1,3-dimethylurea) occurs between two major states (unfolded and compact). However, the free energy of the Caf1 compact structure is only 8.9–9.2 kJ/mol (while for single-domain small proteins it is 21–63 kJ/mol).     

Stabilization energy of the compact Caf1(13-149) subunit from Yersinia pestis

It has been shown by a variety of methods (circular dichroism, viscosimetry, intrinsic fluorescence, and fluorescence of labels) that, as in the case of small globular proteins the folding-unfolding transition in the Caf1(13-149) subunit under the action of two denaturants (urea and 1,3-dimethylurea) occurs between two major states (unfolded and compact). However, the free energy of the compact structure is only 8/9-9/2 kJ/mol (similar values for single-domain small proteins are in the range of 21-63 kJ/mol).     

A protein lacking an essential input to its tertiary structure does fold into a compact globule

It is shown by equilibrium ultracentrifugation, velocity sedimentation, and viscometry that an N-truncated structural protein Caf1 (Cafl_13–149) of the Yersinia pestis capsular antigen fiber exists as a monomer in solution and is capable of folding from denatured state into a compact globular state by itself, without involvement of a chaperone or other subunits. This happens despite the fact that in the norm, important information on the tertiary structure of each Caf1 subunit (specifically, completion of its hydrophobic core) is provided by the “donor” segment Ala1-Thr12 of the neighboring fiber subunit.     

Conserved Hydrophobic Clusters on the Surface of the Caf1A Usher C-Terminal Domain Are Important for F1 Antigen Assembly

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A_C). Caf1A_C is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A_C is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A_C were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems.     

Heightened stability of polcalcin Phl p 7 is correlated with strategic placement of apolar residues

Phl p 7 exhibits atypical conformational stability and a diminutive denaturational heat capacity increment, ΔC_p. Because exposure of apolar surface largely dictates the magnitude of ΔC_p, a depressed value could signify an unusually compact unfolded state. The volume of the denatured state ensemble (DSE) is evidently inversely correlated with mean hydrophobicity [Pace et al., Protein Sci. 19 (2010) 929-943]. Interestingly, apolar residues replace more polar ones at four positions in Phl p 7. We herein examine the consequences of replacing those residues with the corresponding ones from Bra n 1, a related isoform. All four mutations - M4H, L21A, I60T, and C63A - destabilize Phl p 7. Our analysis suggests that the DSE of Phl p 7 is indeed highly compact and that the substitutions act by increasing its volume and solvent-accessibility. All four mutations increase the urea m value; L21A, I60T, and C63A also yield a perceptible increase in ΔC_p.     

The interaction of lysozyme with caffeine,theophylline and theobromine in solution

The interactions of lysozyme with caffeine (Caf), theophylline (Tph) and theobromine (Tbr) were investigated using UV–Vis absorption, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques. The results revealed that Caf (Tph or Tbr) caused the fluorescence quenching of lysozyme by the formation of Caf (Tph or Tbr)–lysozyme complex. The binding constants (K _A) and thermodynamic parameters (ΔG°, ΔH°, ΔS°) at two different temperatures, the binding locality, and the binding power were obtained. The results showed that the process of binding Caf (Tph or Tbr) to lysozyme was a spontaneous molecular interaction procedure and the hydrophobic and electrostatic interactions play a major role in stabilizing the complex; The distance r between donor (lysozyme) and acceptor (Caf, Tph or Tbr) was obtained according to fluorescence resonance energy transfer. The effect of Caf (Tph or Tbr) on the conformation of lysozyme was analyzed using synchronous fluorescence and three-dimensional fluorescence spectra techniques. The results showed that the binding of Caf (Tph or Tbr) to lysozyme induced some micro-environmental and conformational changes in lysozyme and disturbed the environment of the polypeptide of lysozyme.     

A combinatorial biophysical approach; FTSA and SPR for identifying small molecule ligands and PAINs

Biophysical methods have emerged as attractive screening techniques in drug discovery both as primary hit finding methodologies, as in the case of weakly active compounds such as fragments, and as orthogonal methods for hit validation for compounds discovered through conventional biochemical or cellular assays. Here we describe a dual method employing fluorescent thermal shift assay (FTSA), also known as differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR), to interrogate ligands of the kinase p38α as well as several known pan-assay interference compounds (PAINs) such as aggregators, redox cyclers, and fluorescence quenchers. This combinatorial approach allows for independent verification of several biophysical parameters such as K_D, k_on, k_off, ΔG, ΔS, and ΔH, which may further guide chemical development of a ligand series. Affinity values obtained from FTSA curves allow for insight into compound binding compared with reporting shifts in melting temperature. Ligand–p38 interaction data were in good agreement with previous literature. Aggregators and fluorescence quenchers appeared to reduce fluorescence signal in the FTSAs, causing artificially high shifts in T_m values, whereas redox compounds caused either shifts in affinity that did not agree between FTSA and SPR or a depression of FTSA signal.     

Suppression of telomerase activity in leukemic cells by mutant forms of <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis> L-asparaginase

Active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA) (RrA_{+N17, D60K, F61L}, RrA_{+N17, A64V, E67K}, RrA_{+N17, E149R, V150P}, Rr_{AE149R, V150P} and RrA_{E149R, V150P, F151T}) have been obtained by the method of site-directed mutagenesis. The variants RrA_{E149R, V150P, F151T} and RrА_{+N17, E149R, V150P} could decrease reduce expression of the hTERT telomerase subunit and, therefore, activity of telomeres in Jurkat cells, but not in cellular lysates. At the same time, L-asparaginasеs of Escherichia coli, Erwinia carotovora, and Wolinella succinogenes, mutant forms RrА_{+N17, D60K, F61L} and RrА_{+N17, A64V, E67K} did not suppress telomerase activity. It is suggested that some regions in the RrA structure (amino acids residues 146–164, 1–17, 60–67) are responsible for suppression of telomerase activity. The results obtained show that antineoplastic activity of some RrA variants is associated both with reduction of concentration of free L-asparagine, and with decreased expression of the hTERT telomerase subunit; this opens new prospects for antineoplastic therapy.     

Thermodynamics and mechanism of high-pressure deactivation and dissociation of porcine lactic dehydrogenase

Lactic dehydrogenase (LDH) from pig heart and pig skeletal muscle can be reversibly dissociated into monomers at high hydrostatic pressure. The reaction can be quantitatively filled by a reversible consecutive dissociation-unfolding mechanism according to Na = 4M ? 4M* (where N is the native letramer, and M and M* two different conformations of the monomer) (K. Müller, et al., Biophys. Chem. 14 (1981) 101). At P ? 1 kbar, the pressure deactivalion of both isoenzymes (H₄ and M₄) is described by the two-state equilibrium N ? 4M. From the respective equilibrium constant and the temperature and pressure dependence of the change in free energy, the thermodynamic parameters of the dissociation/deactivation may be determined, e.g., for LDH-M₄: Δg_Diss = 110 $\text{kJ}\text{mol}$, ΔSDiss = ?860 J/K per mol, ΔH_Diss = ?124 $\text{kJ}\text{mol}$ (enzyme concentration 10 $\text{μg}\text{ml}$, in Tris-HCl buffer, pH 7.6, I = 0.16 M, 293 K, 0.8 kbar); the dissociation volume is found to be ΔV_Diss = ?420 $\text{ml}\text{mol}$ (0.7 < p < 0.9 kbar). Measurements using 8-anilino-1-naphlhalenesulfonic acid (ANS) as extrinsic fluorophore demonstrate that the occurrence of hydrophobic surface area upon dissociation parallels the decrease in reactivation yield after pressurizarion beyond 1 kbar. Within the range of reversible deactivation (p < 1 kbar) no increase in ANS fluorescence is detectable, thus indicating compensatory effects in the process of subunit dissociation. ²H₂O is found to stabilize the enzyme towards pressure dissociation, in accordance with the involvement of hydrophobic interactions in the subunit contact of both isoenzymes of LDH.     

In vitro studies on the interaction between human serum albumin and fosfomycin disodium salt,an antibiotic drug by multi-spectroscopic and molecular docking methods

The interaction between the human serum albumin (HSA) and drug, fosfomycin disodium salt (FOS) has been studied by different spectroscopic techniques. The experimental results showed a static quenching mechanism in the interaction of FOS with HSA. The number of binding sites, n and observed binding constant K _a were measured by fluorescence quenching method. The thermodynamic parameters ΔG°, ΔH° and ΔS° were calculated according to van’t Hoff equation. The calculated distance r between FOS and the protein is evaluated according to the theory of Förster energy transfer. A change in the secondary structure of the protein was evident from the circular dichroism measurements, synchronous fluorescence and three-dimensional fluorescence spectra.     

Calorimetric determination of thermodynamic parameters of 2′-dUMP binding to Leishmania major dUTPase

We have investigated the binding of 2′-deoxyuridine 5′-monophosphate (2′-dUMP) to Leishmania major deoxyuridine 5′-triphosphate nucleotide hydrolase (dUTPase) by isothermal titration microcalorimetry under different experimental conditions. Binding to dimeric L. major dUTPase is a non-cooperative process, with a stoichiometry of 1 molecule of 2′-dUMP per subunit. The utilization of buffers with different ionization enthalpies has allowed us to conclude that the formation of the 2′-dUMP–dUTPase complex, at pH 7.5 and 30 °C, is accompanied by the uptake of 0.33±0.05 protons per dUTPase subunit from the buffer media. Moreover, 2′-dUMP shows a moderate affinity for the enzyme, and binding is enthalpically driven across the temperature range studied. Besides, whereas ΔG° remains practically invariant as a function of temperature, both ΔH and ΔS° decrease with increasing temperature. The T_S and T_H were 23.4 and 13.6 °C, respectively. The temperature dependence of the enthalpy change yields a heat capacity change of ΔC_p°=?618.1±126.4 cal·mol^?1·K^?1, a value low enough to discard major conformational changes, in agreement with the fitting model. An interpretation of this value in terms of solvent-accessible surface areas is provided.     

The influence of unsaturation on the phase transition temperatures of a series of heteroacid phosphatidylcholines containing twenty-carbon chains

A series of heteroacid sn-1,2 phosphatidylcholines (PC) with twenty-carbon fatty acyl chains has been synthesized. Each PC contained eicosanoate (20:0) in the sn-1 position and one of a group of eicosaenoic acids with increasing numbers of cis double bonds in the sn-2 position. The double bonds were at positions Δ11 (20:1), Δ11,14 (20:2), Δ11,14,17 (20:3), or Δ5,8,11,14 (20:4). The disaturated PC containing two eicosanoate chains was also studied. Aqueous dispersions of these PC were analyzed by differential scanning calorimetry, and data for the gel to liquid-crystalline transitions (given as PC: T_c (° C), T_max (° C), ΔH(kcal/mol)) were as follows - 20:0-20:0 PC: 66.8, 68.4, 15; 20:0-20:1 PC: 19.8, 22.2, 8; 20:0-20:2 PC: −4.3, 1.8, 5; 20:0-20:3 PC: 1.2, 4.4, 7; 20:0-20:4 PC: −10.7, −6.8, 3. Double bonds in excess of two per chain did not substantially change the transition temperatures of these heteroacid PC. There was a small effect of the location of the multiple double bonds on the transition temperature. The data is consistent with the model that the transition temperatures are determined by a balance between a decrease in the packing density in the gel and a decrease in the rotational freedom of the chains in the liquid crystal, both caused by the double bonds ((1983) Biochemistry 22, 1466–1473).     

Reversible unfolding of the β2 subunit of Escherichia coli tryptophan synthetase and its proteolytic fragments

The guanidine hydrochloride-induced reversible unfolding transitions at 4 °C of the β₂ subunit of tryptophan synthetase (l-serine hydrolyase (adding indole) EC. 4.2.1.20) and of its two proteolytic fragments, F₁ and F₂, are compared. The unfolding of the β₂ subunit shows a multistate behaviour, as judged by circular dichroism and fluorescence measurements. When isolated, the two fragments have different stabilities. Within β₂, the region corresponding to the large fragment, F₁ behaves as the corresponding isolated fragment, and no stabilization arising from the interaction with the complementary fragment can be detected. The same behaviour is suggested for the small fragment, F₂. These results lead to the apparent conclusion that, at least under these experimental conditions, the interactions between domains do not contribute greatly to the energetics of the folding process of the large β₂ protein.     

Isokinetic Relationship in the Oxidation of Cuprous Stellacyanin by Cobalt(III) Complexes

Rate parameters have been obtained for the oxidation of cuprous stellacyanin by cobalt(III) ions of the form cis(N)-[CoN₂O₄]^?, including cis(N)-[Co(NTA)(gly)]^?, cis(N)-[Co(IDA)₂]^?, [Co(en)(ox)₂]^?(μ 0.5 M(phosphate), pH 7.0), and Co(EDTA)^?(μ 0.1 M(NaCl), pH 7.2, 0.001 M phosphate). An excellent isokinetic correlation between the activation parameters ΔH^≠ and ΔS^≠ exists for the reactions of aminopolycarboxylatocobalt(III) ions with reduced stellacyanin (β = 300 ± 12 K; correlation coefficient = 0.995). It is concluded that enthalpy-entropy compensation in these reactions may be understood in terms of differing orientations preferred by the various oxidants in forming precursor complexes with the reduced blue protein. While ΔH^≠ and ΔS^≠ values for electron transfer from stellacyanin to cis(N)-[CoN₂O₄]^? ions vary over ranges of 10.7 kcal/mol and 34 cal/mol-deg, respectively, room temperature rate constants are relatively constant (3.6–34.5 M^?1 sec^?1), as expected from Marcus theory for outer sphere electron transfer.     

Thermodynamics of complexation of lanthanides by 3- and 4-hydroxybenzoic acids

The thermodynamic parameters (log β₁₀₁, ΔH₁₀₁, and ΔS₁₀₁) for the formation of the 1:1 complexes between lanthanide cations and 3- and 4-hydroxybenzoate anions were determined by potentiometric and calorimetric titrations in aqueous solutions of 0.10 M (NaClO₄) ionic strength at 25 °C.     

Ice nucleation and supercooling behavior of polymer aqueous solutions

We determined the homogeneous nucleation temperature depression, ΔT_f,hom, the equilibrium melting point depression, ΔT_m, and the value λ, which can be obtained from the linear relationship ΔT_f,hom = λΔT_m, for aqueous solutions of PEG (200-20,000 g mol⁻¹), PVP (10,000, 35,000, 40,000 g mol⁻¹), and dextran (10,000 g mol⁻¹) in the concentration range 0-40 wt% using the emulsion method. The molecular weight dependence of T_f,hom, T_m, and λ in PEG aqueous solutions was found to change in the vicinity of Mw 600-1540 at all concentrations. In addition, it was confirmed that for all of the polymers studied, there was a good linear relationship between λ and the logarithmic value of the self-diffusion coefficient D₀ of the solute molecule. These results indicate that the parameters that describe non-equilibrium freezing, such as T_f,hom and λ, are dependent on solution properties such as viscosity and self-diffusion of solute molecules.     

Solution properties of synthetic polypeptides. XII. Enthalpy changes accompanying helix-coil transition of polypeptide

For helix-coil transitions of polypeptide in binary mixtures consisting of helix-forming solvent and coil solvent, the transition enthalpy ΔH(T,x) has been found to depend significantly on temperature (T) and solvent composition (x). For such systems, calorimetric measurements may yield some averages of ΔH(T,x) which are no longer amenable to direct comparison with ΔH itself. Theoretical equations relating calorimetric data to ΔH(T,x) are derived and tested favorably with experimental data. It is demonstrated that the transition enthaply from heat capacity measurements is approximately equal to ΔH_cfm, while those from heat of dilution and heat of solution measurements are equal to ΔH_c. Here ΔH_c denotes the value of ΔH at the transition point and f_m represents the maximum helical content attained in a thermally induced transition. The discrepancies among calorimetric data are also discussed.     

Magneto-structural correlations in chains of bifolded Cu2Cl62− dimers

The magnetic susceptibility of two ACuCl₃ salts containing chains of bibridged Cu₂Cl₆²⁻ dimers has been measured. Each copper ion bridges to a chloride in an adjacent dimer, thus assuming a 4 + 1 coordination geometry. Structurally, each dimer is characterized by a bridging CuClCu angle, ø, and a fold angle, σ, at each copper center. The high temperature susceptibility data for (4-benzylpiperidinium)CuCl₃ (ø=95.3°, σ=28.9°) obey a Curie-Weiss law which has a positive intercept (θ=13 K), indicating a predominant ferromagnetic interaction. For the low temperature data, the Curie-Weiss plot has a negative intercept (θ′=−4 K), showing that a net antiferromagnetic coupling exists. The data is interpreted quantitatively in terms of a system of ground state triplet dimers, with singlet-triplet energy splitting ΔE(=2J) of ΔE/k=60 K with interdimer interactions, accounted for with a mean field approximation of J′/k=−3 K. The salt (paraquat)Cu₂Cl₆ (ø=97.5°, σ=31.7°) behaves as an antiferromagnetic alternating chain with J/k=19 K and J′/k=2 K. The magnetic properties of two other salts containing structurally similar chains, (CH₃)₂NH₂CuCl₃ and (CH₃)₂CHNH₃CuCl₃, have been reinvestigated using pulsed high field magnetization techniques. Comparison of these systems shows that, with ø constant, there is an approximate linear relationship between ΔE and the fold angle, σ. At ø=95.5°±0.3°, the interaction is ferromagnetic for σ>22° and antiferromagnetic for smaller values of σ. These conclusions are confirmed by extended Hückel MO calculations for the intradimer interactions within the Hoffmann framework for exchange coupling.     

Effects of body size and environment on diet-tissue δC fractionation in fishes

δ¹³C data are often used in trophodynamic research where diet-tissue fractionation (Δδ¹³C) is assumed to be 0-1‰ per trophic level and unaffected by the size of animals or their environment. Variation in Δδ¹³C will influence conclusions about food sources, energy pathways and trophic level. To assess the effects of body size, age and environmental conditions on Δδ¹³C, European sea bass (Dicentrarchus labrax) were reared on constant diets of dab (Limanda limanda) or (Ammodytes marinus) for 2years under natural environmental regimes. Bass were sampled approximately monthly to determine Δδ¹³C for muscle, heart and liver tissue and were 1.66‰, − 0.18‰, − 1.77‰ (sandeel diet) and 1.34‰, − 1.18‰, − 1.75‰ (dab diet) respectively. Arithmetic lipid correction increased Δδ¹³C to > 2‰ for muscle and liver. Δδ¹³C was dependent on body mass and experimental duration (age) and generally declined with weight or time even after correction for lipid content. For liver, increasing temperature increased Δδ¹³C. The Δδ¹³C estimates from this study were compared with all available published Δδ¹³C estimates for fish. Bass muscle Δδ¹³C was similar to previous estimates for fish white muscle Δδ¹³C (1.56 ± 1.10‰) and whole body Δδ¹³C (1.52 ± 1.13‰). Fractionations derived in this study, combined with those from the literature, support the use of diet-tissue fractionation values of between 1‰-2‰ for δ¹³C, rather than the commonly used 0‰ − 1‰. For muscle Δδ¹³C, 1.5‰ is appropriate.     

Chiral metal complexes. Part 22. Stereoisomers of the dichloro [3,6-diaza-2S,7S-di(2-pyridyl)octane] - cobalt(III) ion and related species

The reaction of 3,6-diaza-2S,7S-di(2-pyridyl)- octane, S,S-peaen, with Co(II) and O₂ in aqueous solution yields a mixture, from which may be isolated three chelate diastereomers after the addition of HCl and HClO₄. These are δ-α-[Co(S,S-peaen)Cl₂]- ClO₄ and its Λ-α and Δ-β analogues. Previous workers had reported that a second β-diastereoisomer could be obtained but it has been shown that this is in fact an isomeric mixture of both Δ-α- and Λ-α- [Co(S,S-peaen)Cl₂]ClO₄. All three isomers react with oxalate anions to form an apparently unique product Λ-α-[Co(S,S-peaen)ox]⁺ in aqueous solution and which can be crystallized as its perchlorate salt. Two of the reactions reported represent unusual examples of octahedral inversions.