Molecular dynamics simulation of the calmodulin-trifluoperazine complex in aqueous solution

Trifluoperazine (TFP) has been widely studied in relation to its mode of binding and its inactivation of calmodulin (CaM). Most studies in solution have indicated that CaM has two high-affinity binding sites for TFP. The crystal structure of the 1:4 CaM-TFP complex (CaM-4TFP) shows that three TFP molecules bind to the C-domain of CaM, and that one TFP molecule binds to the N-domain. In contrast, the crystal structure of the 1:1 CaM-TFP complex (CaM-1TFP) shows that one TFP molecule binds to the C-domain. It has been thought that the binding of one TFP molecule to the C-domain is followed by binding to the N-domain. The crystal structure of the 1:2 CaM-TFP complex (CaM-2TFP), moreover, has recently been determined, showing that two TFP molecules bind to the C-domain. In order to determine the structure of the CaM-TFP complex and to clarify the interaction between CaM and TFP in solution, we performed a molecular dynamics simulation of the CaM-TFP complex in aqueous solution starting from the CaM-4TFP crystal structure. The obtained solution structure is very similar to the CaM-2TFP crystal structure. The computer simulation showed that the binding ability of the secondary binding site of the C-domain is higher than that of the primary binding site of the N-domain.     

A structural and thermodynamic analysis of novatrone and flavin mononucleotide heteroassociation in aqueous solution by 1H NMR spectroscopy

A heteroassociation of antitumor antibiotic novatrone (NOV) and flavin mononucleotide (FMN) in aqueous solution was studied by one- and two-dimentional 1H NMR spectroscopy (500 MHz) to elucidate the molecular mechanism of the possible combined action of the antibiotic and vitamin. The equilibrium reaction constants, induced proton chemical shifts, and the thermodynamic parameters (deltaH and deltaS) of the NOV and FMN heteroassociation were determined from the concentration and temperature dependences of proton chemical shifts of the aromatic molecules. The most favorable structure of the 1 : 1 NOV-FMN complex was determined by both the method of molecular mechanics (X-PLOR software) and the induced proton chemical shifts of the molecules. An analysis of the results suggests that the NOV-FMN intermolecular complexes are mainly stabilized by stacking interactions of their aromatic chromophores. An additional stabilization is possible due to intermolecular hydrogen bonds. It was concluded that the aromatic molecules of vitamins, in particular, FMN, can form energetically favorable heterocomplexes with aromatic antitumor antibiotics in aqueous solutions, which could result in a modulation of their medical and biological action.     

Kinetic and thermodynamic analysis of thermal unfolding of recombinant erythropoietin

Thermal stress was used to assess the stability of recombinant human erythropoietin (EPO) derived from Chinese hamster ovary cells. In 20 mm phosphate at pH 7.0, this protein had a highly reversible thermal unfolding as observed by far UV circular dichroism (CD) and native gel analysis, with no indication of protein aggregation. It had a relatively low melting temperature at 53 degrees C. Assuming a two-state transition, the observed reversibility permits thermodynamic analysis of the unfolding of EPO, which shows that the free energy of unfolding at 25 degrees C is only 6-7 kcal/mol. Upon heating to 79 degrees C over 30 min, however, this protein does undergo aggregation as assessed by native gel. In 20 mm phosphate and citrate at pH 7.0, the results are similar, i.e., EPO suffered a substantial aggregation, while it showed little aggregation in 20 mm Tris or histidine at pH 7.0 and 20 mm glycine at pH 6.3 under identical heat treatment.     

Kinetic studies of the thermal decomposition of 2-chloroethylphosphonic Acid in aqueous solution

The decomposition of 2-chloroethylphosphonic acid in aqueous solution has been studied at pH values from 6 to 9 and at temperatures in the 30 to 55 C range. The rate of decomposition is estimated from the rate of formation of ethylene. The rate is proportional to the concentration of the phosphonate dianion and is independent of the hydroxyl ion concentration. The rate constant at 40 C is 1.9 × 10⁻⁴ sec⁻¹ and the activation energy is 29.8 kcal mol⁻¹. The rate of reaction is not affected significantly by the presence of potassium iodide or urea (substances which increase the rate of leaf abscission in trees sprayed by 2-chloroethylphosphonic acid). The rate decreases slightly in the presence of low concentrations of magnesium and calcium ions.     

Mercury removal from aqueous solution by dried biomass of indigenous Vibrio parahaemolyticus PG02: Kinetic,equilibrium, and thermodynamic studies

In this study, for the first time the potential use of dried Vibrio parahaemolyticus PG02 to remove mercury from synthetic effluent was investigated by considering equilibrium, kinetic, and thermodynamic aspects. The results indicated that Hg²⁺ biosorption was best described by the pseudo-second order model. In addition, it was found that intraparticle diffusion was not the sole rate-limiting step. The ion-exchange mechanism was a predominant biosorption mechanism in the first 15 min of contact. The Langmuir isotherm better described the equilibrium data of Hg²⁺ biosorption than the Freundlich isotherm. According to this model, the maximum biosorption capacity was found to be 9.63 × 10⁻⁴ mol g⁻¹ at optimum conditions (pH = 6.0 and temperature =35 °C).     

Kinetic analysis of the RNAi enzyme complex

The siRNA-directed ribonucleoprotein complex, RISC, catalyzes target RNA cleavage in the RNA interference pathway. Here, we show that siRNA-programmed RISC is a classical Michaelis-Menten enzyme in the presence of ATP. In the absence of ATP, the rate of multiple rounds of catalysis is limited by release of the cleaved products from the enzyme. Kinetic analysis suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage, and product release. Bases near the siRNA 5' end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3' regions of the siRNA provide a helical geometry required for catalysis. Finally, the position of the scissile phosphate on the target RNA seems to be determined during RISC assembly, before the siRNA encounters its RNA target.     

Kinetic and thermodynamic analysis of the interaction of cations with dialkylglycine decarboxylase

Dialkylglycine decarboxylase (DGD) is a tetrameric pyridoxal phosphate (PLP)-dependent enzyme that catalyzes both decarboxylation and transamination in its normal catalytic cycle. Its activity is dependent on cations. Metal-free DGD and DGD complexes with seven monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) and three divalent cations (Mg(2+), Ca(2+), and Ba(2+)) have been studied. The catalytic rate constants for cation-bound enzyme (ck(cat) and ck(cat)/bK(AIB)) are cation-size-dependent, K(+) being the monovalent cation with the optimal size for catalytic activity. The divalent alkaline earth cations (Mg(2+), Ca(2+), and Ba(2+)) all give approximately 10-fold lower activity compared to monovalent alkali cations of similar ionic radius. The Michaelis constant for aminoisobutyrate (AIB) binding to DGD-PLP complexes with cations (bK(AIB)) varies with ionic radius. The larger cations (K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) give smaller bK(AIB) ( approximately 4 mM), while smaller cations (Li(+), Na(+)) give larger values (approximately 10 mM). Cation size and charge dependence is also found with the dissociation constant for PLP binding to DGD-cation complexes (aK(PLP)). K(+) and Rb(+) possess the optimal ionic radius, giving the lowest values of aK(PLP). The divalent alkaline earth cations give aK(PLP) values approximately 10-fold higher than alkali cations of similar ionic radius. The cation dissociation constant for DGD-PLP-AIB-cation complexes (betaK(M)z+) was determined and also shown to be cation-size-dependent, K(+) and Rb(+) yielding the lowest values. The kinetics of PLP association and dissociation from metal-free DGD and its complexes with cations (Na(+), K(+), and Ba(2+)) were analyzed. All three cations tested increase PLP association and decrease PLP dissociation rate constants. Kinetic studies of cation binding show saturation kinetics for the association reaction. The half-life for association with saturating Rb(+) is approximately 24 s, while the half-life for dissociation of Rb(+) from the DGD-PLP-AIB-Rb(+) complex is approximately 12 min.     

Structural and thermodynamic analysis of the GFP:GFP-nanobody complex

The green fluorescent protein (GFP)-nanobody is a single-chain VHH antibody domain developed with specific binding activity against GFP and is emerging as a powerful tool for isolation and cellular engineering of fluorescent protein fusions in many different fields of biological research. Using X-ray crystallography and isothermal titration calorimetry, we determine the molecular details of GFP:GFP-nanobody complex formation and explain the basis of high affinity and at the same time high specificity of protein binding. Although the GFP-nanobody can also bind YFP, it cannot bind the closely related CFP or other fluorescent proteins from the mFruit series. CFP differs from GFP only within the central chromophore and at one surface amino acid position, which lies in the binding interface. Using this information, we have engineered a CFP variant (I146N) that is also able to bind the GFP-nanobody with high affinity, thus extending the toolbox of genetically encoded fluorescent probes that can be isolated using the GFP-nanobody.     

Kinetic and thermodynamic analysis of the interactions of 23-residue peptides with endotoxin

Many naturally occurring peptides exhibit lipopolysaccharide binding properties. In this work we describe the endotoxin binding properties of a series of 23-residue peptides based on the sequence corresponding to the antisense strand of the magainin gene. Biochemical and biophysical characterization of these peptides reveals that they have the tendency to perturb both the inner and outer membranes of test pathogens. Structurally these peptides are amphiphilic and adopt helical conformations in membranes. Three of the seven peptides tested have high affinities for endotoxin that approach the values shown by polymyxin B, a cyclic cationic acylated decapeptide, which is used clinically in treating extreme cases of sepsis. The kinetic parameters obtained using stopped-flow methods and BIAcore analysis, when considered in conjunction with the isothermal titration calorimetry-derived thermodynamic parameters, allow us to highlight the key structural features essential for lipopolysaccharide (LPS) recognition by these peptides. The studies stress the role of ionic forces in the initial recognition of LPS. The fortification of the strength of these ionic charges increases affinity for LPS, whereas the hydrophobic residues involved in interactions are more amenable to disruptions in contiguity. Peptides that improve these features further are expected to perform better as endotoxin-neutralizing agents.     

Conformational analysis of the tetrapeptide Pro-D-Phe-Pro-Gly in aqueous solution

Conformational investigations of the tetrapeptide Pro-D-Phe-Pro-Gly in water solution were carried out by 1H and 13C NMR spectroscopy. The internal proline residue allows for the possibility of cis/trans isomerization about the D-Phe-Pro peptide bond resulting in two conformational isomers. The major isomer was identified as the trans isomer. The pH-dependence of the cis/trans equilibrium supports an additional stabilisation of the trans isomer by an intramolecular ionic interaction between the amino- and carboxy-terminus in the zwitterionic state. Based on 13C spin-lattice relaxation times (T1), different pyrrolidine ring conformations of Pro1 and Pro3 could be determined. By combination of several NMR data (vicinal coupling constants 3JN alpha, temperature dependence of the NH chemical shifts, differences in the chemical shifts between the beta and gamma carbons of the proline residues) and energy minimization calculations, a type II' beta-turn should contribute considerably to the overall structure of the trans isomer.     

Kinetic analysis of the interleukin-13 receptor complex

Interleukin (IL)-13 is a key cytokine associated with the asthmatic phenotype. It signals via its cognate receptor, a complex of IL-13 receptor alpha1 chain (IL-13Ralpha1) with IL-4Ralpha; however, a second protein, IL-13Ralpha2, also binds IL-13. To determine the binding contributions of the individual components of the IL-13 receptor to IL-13, we have employed surface plasmon resonance and equilibrium binding assays to investigate the ligand binding characteristics of shIL-13Ralpha1, shIL-13Ralpha2, and IL-4Ralpha. shIL-13Ralpha1 bound IL-13 with moderate affinity (K(D) = 37.8 +/- 1.8 nm, n = 10), whereas no binding was observed for hIL-4Ralpha. In contrast, shIL-13Ralpha2 produced a high affinity interaction with IL-13 (K(D) = 2.49 +/- 0.94 nm n = 10). IL-13Ralpha2 exhibited the binding characteristics of a negative regulator with a fast association rate and an exceptional slow dissociation rate. Although IL-13 interacted weakly with IL-4Ralpha on its own (K(D) > 50 microm), the presence of hIL-4Ralpha significantly increased the affinity of shIL-13Ralpha1 for IL-13 but had no effect on the binding affinity of IL-13Ralpha2. Detailed kinetic analyses of the binding properties of the heteromeric complexes suggested a sequential mechanism for the binding of IL-13 to its signaling receptor, in which IL-13 first binds to IL-13Ralpha1 and this then recruits IL-4Ralpha to stabilize a high affinity interaction.     

Effects of pH and temperature on the structural and thermodynamic character of alpha-syn12 peptide in aqueous solution

The structural and thermodynamic characters of alpha-syn12 peptide in aqueous solution at different pH and temperatures have been investigated through temperature replica exchange molecular dynamics (T-REMD) simulations with GROMOS 43A1 force field. The two independent T-REMD simulations were completed at pH = 7.0 and 10.0, respectively. Each replica was run for 300 ns. The structural and thermodynamic characters of alpha syn12 peptide were studied based on the distributions of backbone dihedral angles, the free energy surface, and the stability of different type structure and the favorite conformations of the peptide. The results showed that the simulation at pH = 10.0 produced more sampling in alpha region than the simulation at pH = 7.0. The temperature changes from 283 K to 308 K result in negligible effects on the distributions of backbone dihedral angle. The beta hairpin conformation with Turn(9-6) and four hydrogen bonds (HB(4-11), HB(6-9), HB(9-6) and HB(11-4)) is the lowest free energy state in the simulation at pH = 7.0. However, for the simulation at pH = 10.0, the lowest free energy state corresponds to a structure with Turn(9-6) and two hydrogen bonds (HB(6-10) and HB(10-6)) induced by an overly strong residue-residue interaction effect between lysine residues. For the simulation at pH = 7.0, the free energy change of the alpha-syn12 peptide from the unfolded state to the beta hairpin state was in good agreement with the experiments and molecular dynamics simulation results for the other beta-peptides, the beta hairpin state of the alpha-syn12 peptide included the conformations that not only the Turn(9-6) is formed, but also the terminus are closed together in space. However, the subtle balances between lysine-lysine interactions and lysine-solvent interaction are disrupted in the simulation at pH = 10.0, which induced the assembly of lysine residues, the beta hairpin conformation is destabilized by the deprotonation of the Lys side chain. This study can help us to understand the conformation changes and the thermodynamic character of alpha;-syn12 peptide at atomic level induced by changing pH and temperature, which is propitious to reveal the nosogenesis of Parkinson disease. In our knowledge, this is the first report to study the influence of pH and temperature on isolated alpha-syn12 peptide in water by T-REMD.     

Spatial structure of angiotensin in an aqueous solution

The spatial structure of spin-labeled angiotensin in aqueous solution wa investigated with the combined use of NMR, fluorescence spectroscopy and energy calculation including Monte-Carlo techniques. The calculated mean values of molecular parameters were compared with the experimental ones. The calculated and experimental mean values were regarded as statistically indistinguishable when the corresponding mean values occurred within the 95% confidence limit. The experimental parameters were shown to be adequately described by calculated conformers only with the assumption of the existence of dynamic equilibrium of conformers in solution. The mean values of statistical weights and their limits providing the agreement between the calculated and experimental data were determined. Two geometrically different forms of backbone structure for C-terminal hexapeptide in aqueous solution were revealed using the discussed approach; the N-terminal part of the molecule appeared to be much more conformationally labile. The model of molecule spatial structure is consistent with available literature data upon angiotensin titration experiments, its complexing with heavy metal ions etc.     

Photostability of lycopene dispersed in an aqueous solution

Lycopene dispersed in aqueous solutions with different dissolved oxygen contents was photo-irradiated by using a xenon weather meter, and the contents of lycopene and dissolved oxygen were measured. Both the degradation of lycopene and the consumption of dissolved oxygen followed a first-order kinetics model. There was a proportional relationship between the degradation content of lycopene and the consumption of dissolved oxygen. These results indicate that dissolved oxygen would also be involved in the photolysis of lycopene.     

Reversible oxygenation of protoheme-imidazole complex in aqueous solution (1,2)

Solutions of protohemin in aqueous buffer containing imidazole were reduced and exposed to carbon monoxide forming the carbon monoxide-imidazole complex similar to that in carboxyhemoglobin. This complex is stable for long periods in the presence of low pressures of oxygen and thus the standard flash photolysis methods can be used to determine rates of combination of the heme-imidazole complex with oxygen. Combination rates for both carbon monoxide and oxygen are faster than any on rates for hemoglobin and oxygen dissociation rates are also faster. But the equilibrium constant for binding of this isolated site is larger than that for hemoglobin.     

Quantitative characterization of the concentration-dependent interaction between molecules of Dextran 70 in aqueous solution: Measurement and analysis in the context of thermodynamic and compressible sphere models

The static light scattering and sedimentation equilibrium of solutions of Dextran 70 were measured as functions of concentration up to 100 g/L in pH 7.4 phosphate-buffered saline at temperatures between 5 and 37 °C. The concentration dependence of scattering intensity and the apparent molar mass obtained from sedimentation equilibrium were found to be nearly independent of temperature over this range to within the uncertainty of measurement. Global analysis of the concentration dependence of both properties yielded a reliable estimate of the concentration-dependent thermodynamic activity coefficient, a quantitative measure of the free energy of self-interaction. The self-interaction between Dextran molecules is compared with that of a globular protein (BSA) and a highly crosslinked polymer of similar molar mass (Ficoll 70). The observed concentration dependence of the free energy of Dextran self-interaction may be quantitatively accounted for by a semi-empirical model in which the polymer molecule is represented by a compressible sphere.     

A thermodynamic and structural analysis of DNA minor-groove complex formation

As part of an effort to develop a better understanding of the structural and thermodynamic principles of DNA minor groove recognition, we have investigated complexes of three diphenylfuran dications with the d(CGCGAATTCGCG)(2) duplex. The parent compound, furamidine (DB75), has two amidine substituents while DB244 has cyclopentyl amidine substituents and DB226 has 3-pentyl amidines. The structure for the DB244-DNA complex is reported here and is compared to the structure of the DB75 complex. Crystals were not obtained with DB226 but information from the DB75 and DB244 structures as well as previous NMR results on DB226 indicate that all three compounds bind in the minor groove at the AATT site of the duplex. DB244 and DB75 penetrate to the floor of the groove and form hydrogen bonds with T8 on one strand and T20 on the opposite strand while DB226 forms a complex with fewer interactions. Binding studies by surface plasmon resonance (SPR) yield -delta G degrees values in the order DB244>DB75>DB226 that are relatively constant with temperature. The equilibrium binding constants for DB244 are 10-20 times greater than that for DB226. Isothermal titration calorimetric (ITC) experiments indicate that, in contrast to delta G degrees, delta H degrees varies considerably with temperature to yield large negative delta Cp degrees values. The thermodynamic results, analyzed in terms of structures of the DNA complexes, provide an explanation of why DB244 binds more strongly to DNA than DB75, while DB266 binds more weakly. All three compounds have a major contribution to binding from hydrophobic interactions but the hydrophobic term is most favorable for DB244. DB244 also has strong contributions from molecular interactions in its DNA complex and all of these factors combine to give it the largest-delta G degrees for binding. Although the factors that influence the energetics of minor groove interactions are varied and complex, results from the literature coupled with those on the furan derivatives indicate that there are some common characteristics for minor groove recognition by unfused heterocyclic cations that can be used in molecular design.