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PubMed2003

Energetics and self-assembly of amphipathic peptide pores in lipid membranes.

We present a theoretical study of the energetics, equilibrium size, and size distribution of membrane pores composed of electrically charged amphipathic peptides. The peptides are modeled as cylinders (mimicking alpha-helices) carrying different amounts of charge, with the charge being uniformly distributed over a hydrophilic face, defined by the angle subtended by polar amino acid residues. The free energy of a pore of a given radius, R, and a given number of peptides, s, is expressed as a sum of the peptides' electrostatic charging energy (calculated using Poisson-Boltzmann theory), and the lipid-perturbation energy associated with the formation of a membrane rim (which we model as being semitoroidal) in the gap between neighboring peptides. A simple phenomenological model is used to calculate the membrane perturbation energy. The balance between the opposing forces (namely, the radial free energy derivatives) associated with the electrostatic free energy that favors large R, and the membrane perturbation term that favors small R, dictates the equilibrium properties of the pore. Systematic calculations are reported for circular pores composed of various numbers of peptides, carrying different amounts of charge (1-6 elementary, positive charges) and characterized by different polar angles. We find that the optimal R's, for all (except, possibly, very weakly) charged peptides conform to the "toroidal" pore model, whereby a membrane rim larger than approximately 1 nm intervenes between neighboring peptides. Only weakly charged peptides are likely to form "barrel-stave" pores where the peptides essentially touch one another. Treating pore formation as a two-dimensional self-assembly phenomenon, a simple statistical thermodynamic model is formulated and used to calculate pore size distributions. We find that the average pore size and size polydispersity increase with peptide charge and with the amphipathic polar angle. We also argue that the transition of peptides from the adsorbed to the inserted (membrane pore) state is cooperative and thus occurs rather abruptly upon a change in ambient conditions.

Cell Membrane Permeability↗
PubMed1989

Interaction of non-piliated Neisseria gonorrhoeae strain 7122 and protein IA with an epithelial cell monolayer.

Studies with [14C] uracil-labelled bacteria revealed that the interaction of Neisseria gonorrhoeae with epithelial cells occurred in a time-dependent reaction which is slightly pH-dependent and optimal at pH 6.5. Immunofluorescence tests and immunoelectron microscopy of ultrathin sections confirmed the attachment of these bacteria to the epithelial cell membrane. The interaction of purified protein I with epithelial cells was time-dependent and reached equilibrium after four hours as shown by tracer experiments with 125I-labeled protein I. Cleavage experiments with trypsin followed by SDS-PAGE and autoradiography indicated that protein I (labeled with 125I) was associated with the membrane of the epithelial cells and only partly accessible by trypsin after its interaction with these mammalian cells. Immunofluorescence tests as well as immunoelectron microscopy with the monoclonal antibody G7A2C and gold-labeled protein A confirmed a dense association pattern of protein I with the cell monolayer.

Antibodies, Monoclonal↗
PubMed1987

Yeast diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase behaves as a dinucleoside tetraphosphate synthetase.

The diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase (Ap4A phosphorylase), recently observed in yeast [Guaranowski, A., & Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547], is shown to be capable of catalyzing the synthesis of Ap4A from ATP + ADP, i.e., the reverse reaction of the phosphorolysis of Ap4A. The synthesis of Ap4A markedly depends on the presence of a divalent cation (Ca2+, Mn2+, or Mg2+). In vitro, the equilibrium constant K = ([Ap4A][Pi])/[(ATP][ADP]) is very sensitive to pH. Ap4A synthesis is favored at low pH, in agreement with the consumption of one to two protons when ATP + ADP are converted into Ap4A and phosphate. Optimal activity is found at pH 5.9. At pH 7.0 and in the presence of Ca2+, the Vm for Ap4A synthesis is 7.4 s-1 (37 degrees C). Ap4A phosphorylase is, therefore, a valuable candidate for the production of Ap4A in vivo. Ap4A phosphorylase is also capable of producing various Np4N' molecules from NTP and N'DP. The NTP site is specific for purine ribonucleotides (N = A, G), whereas the N'DP site has a broader specificity (N' = A, C, G, U, dA). This finding suggests that the Gp4N' nucleotides, as well as the Ap4N' ones, could occur in yeast cells.

Acid Anhydride Hydrolases↗
PubMed1999

Why Are Copper(II) Amino Acid Complexes Not Planar in Their Crystal Structures? An ab Initio and Molecular Mechanics Study.

This paper presents geometries of copper(II) chelates with L-alanine, L-leucine, and L-N,N-dimethylvaline optimized by the hybrid density functional method B3LYP. According to the molecular quantum mechanics results, a square-planar copper(II) coordination geometry is electronically favored in vacuo. Deviations from the planar configuration observed in the crystal state should be attributed to sterical intramolecular and/or intermolecular effects. This paper proposes a new molecular mechanics model for tetracoordinated copper(II) amino acidates to investigate these effects in detail. The empirical parameter set for the selected potential energy functions was optimized both with respect to the X-ray crystal structures (internal coordinates and unit cell constants) and with respect to the quantum mechanically derived valence angles around copper. To test this newly developed force field (FF), the equilibrium geometries of 10 molecules are predicted in vacuo and in approximate crystalline surrounding. The results were compared with their ab initio and experimental crystal structures, respectively. The unit cell volumes were reproduced in a range from -7.0% to 2.1%. The total root-mean-square deviations between the experimental and FF in crystal internal coordinates were 0.017 Å in the bond lengths, 2.2 degrees in the valence angles, and 3.6 degrees in the torsion angles. The force field is capable of reproducing the changes in the chelate rings' torsion angles caused by the crystal packing forces and successfully explains the nonplanarity of Cu(II) amino acid complexes in their crystal structures.

Journal Article↗
PubMedDate not supplied

[Kinetics of the reaction between formaldehyde and proteins].

The reaction between formaldehyde and protein has been studied with the use of a radioactive indicator. In the presence of excess formaldehyde the reaction is kinetically of the first order. The accordance of experimental results to the kinetic scheme of a biphasic process was shown. On this basis experimental and veritable rate constants, equilibrium constants of mono- and dimetilol derivatives formation, the energy of activation and pseudothermodynamic parameters were determined. The dependence of reaction rate and degree of protein modification from formaldehyde concentration and temperature are discussed. Given results permit to optimize and control the process of interaction between formaldehyde and proteins.

Formaldehyde↗
PubMed1998

Limiting models for calcification in fibrous tissues adjacent to orthopedic implants: variational indicator functions and influences of implant stiffness.

Calcification and eventual integration of orthopedic implants into bone is important to many load-bearing devices, and the influence of load and implant stiffness on this process are assessed in this mathematical modelling study. Three research questions are posed in this study. First, can limiting material models provide useful information on the overall behavior of the tissue adjacent to a loaded orthopedic implant? Second, can the limiting models lead to optimization criteria? Third, can an optimization approach be used to differentiate between the four prospective remodeling rate equations which are proposed? The answers are yes, yes, and no, respectively. A two degree of freedom lumped parameter model for axial loading of an intramedullary implant is considered. Two limiting composite material models are used, and the strain energy density in the calcified and non-calcified phases are assessed as stimuli for calcification. The rate equations posed here assume that the calcified material volume fraction decreases at high strain-energy densities, and increases at small strain-energy densities. In all four cases (both models, both phases) the steady states for these rate equations find equilibrium points of indicator functions which are a weighted sum of total strain energy and the mass of calcified tissue in the layer considered. The weights on strain-energy density and mass differ in each case. This shows that for appropriate choices of parameters, all four models can yield the same results, and it also shows that an optimization approach does not uniquely determine the appropriate rate equation in these cases. The rate equations showed complicated dynamic behavior and a phase-plane analysis was used which led to upper bounds on load, which depended on implant stiffness and distal support. The predictions of the four cases studied are compared.

Bone and Bones↗
PubMed2003

Fast gradient HPLC method to determine compounds binding to human serum albumin. Relationships with octanol/water and immobilized artificial membrane lipophilicity.

A fast gradient HPLC method (cycle time 15 min) has been developed to determine Human Serum Albumin (HSA) binding of discovery compounds using chemically bonded protein stationary phases. The HSA binding values were derived from the gradient retention times that were converted to the logarithm of the equilibrium constants (logK HSA) using data from a calibration set of molecules. The method has been validated using literature plasma protein binding data of 68 known drug molecules. The method is fully automated, and has been used for lead optimization in more than 20 company projects. The HSA binding data obtained for more than 4000 compounds were suitable to set up global and project specific quantitative structure binding relationships that helped compound design in early drug discovery. The obtained HSA binding of known drug molecules were compared to the Immobilized Artificial Membrane binding data (CHI IAM) obtained by our previously described HPLC-based method. The solvation equation approach has been used to characterize the normal binding ability of HSA, and this relationship shows that compound lipophilicity is a significant factor. It was found that the selectivity of the "baseline" lipophilicity governing HSA binding, membrane interaction, and octanol/water partition are very similar. However, the effect of the presence of positive or negative charges have very different effects. It was found that negatively charged compounds bind more strongly to HSA than it would be expected from the lipophilicity of the ionized species at pH 7.4. Several compounds showed stronger HSA binding than can be expected from their lipophilicity alone, and comparison between predicted and experimental binding affinity allows the identification of compounds that have good complementarities with any of the known binding sites.

Blood Proteins↗
PubMed2006

Natural selection for kinetic stability is a likely origin of correlations between mutational effects on protein energetics and frequencies of amino acid occurrences in sequence alignments.

It appears plausible that natural selection constrains, to some extent at least, the stability in many natural proteins. If, during protein evolution, stability fluctuates within a comparatively narrow range, then mutations are expected to be fixed with frequencies that reflect mutational effects on stability. Indeed, we recently reported a robust correlation between the effect of 27 conservative mutations on the thermodynamic stability (unfolding free energy) of Escherichia coli thioredoxin and the frequencies of residues occurrences in sequence alignments. We show here that this correlation likely implies a lower limit to thermodynamic stability of only a few kJ/mol below the unfolding free energy of the wild-type (WT) protein. We suggest, therefore, that the correlation does not reflect natural selection of thermodynamic stability by itself, but of some other factor which is linked to thermodynamic stability for the mutations under study. We propose that this other factor is the kinetic stability of thioredoxin in vivo, since( i) kinetic stability relates to irreversible denaturation, (ii) the rate of irreversible denaturation in a crowded cellular environment (or in a harsh extracellular environment) is probably determined by the rate of unfolding, and (iii) the half-life for unfolding changes in an exponential manner with activation free energy and, consequently, comparatively small free energy effects can have deleterious consequences for kinetic stability. This proposal is supported by the results of a kinetic study of the WT form and the 27 single-mutant variants of E. coli thioredoxin based on the global analyses of chevron plots and equilibrium unfolding profiles determined from double-jump unfolding assays. This kinetic study suggests, furthermore, one of the factors that may contribute to the high activation free energy for unfolding in thioredoxin (required for kinetic stability), namely the energetic optimization of native-state residue environments in regions, which become disrupted in the transition state for unfolding.

Amino Acid Sequence↗
PubMed2005

Cys(x)His(y)-Zn2+ interactions: possibilities and limitations of a simple pairwise force field.

In zinc proteins, the Zn2+ cation frequently binds with a tetrahedral coordination to cysteine and histidine side chains. We examine the possibilities and limitations of a classical, pairwise force field for molecular dynamics of such systems. Hartree Fock and density functional calculations are used to obtain geometries, charge distributions, and association energies of side chain analogues bound to Zn2+. Both ionized and neutral cysteines are considered. Two parameterizations are obtained, then tested and compared through molecular dynamics simulations of two small, homologous proteins in explicit solvent: Protein Kinase C and the Cysteine Rich Domain (CRD) of Raf, which have two Cys3His-Zn2+ groups each. The lack of explicit polarizability and charge transfer in the force field leads to poor accuracy for the association energies, and to parameters--including the zinc charge, that depend on the number of bound cysteines and their protonation state. Nevertheless, the structures sampled with the best parameterization are in good overall agreement with experiment, and have zinc coordination geometries compatible with related structures in the Cambridge Structural Database and the Protein Data Bank. Non-optimized parameters lead to poorer structures. This suggests that while a simple force field is not appropriate for processes involving exchange between water and amino acids in the zinc coordination sphere (e.g. protein unfolding), it can be useful for equilibrium simulations of stable Cys3His zinc fingers.

Computer Simulation↗
PubMed2000

Effects of high hydrostatic pressure on membrane proteins of Salmonella typhimurium.

Salmonella typhimurium is a leading cause of foodborne diseases. Today high hydrostatic pressure treatments are considered as alternative methods of preservation. To select optimal conditions of treatment, we have to characterize the cell targets of pressure. In this study the action of pressure on the bacterial membrane proteins is analysed. The total membrane extract is obtained by lysis of cells separated by equilibrium density gradient centrifugation. Protein content is analysed by electrophoresis SDS-PAGE and visualised by silver stain. Electrophoretic profiles reveal the presence of three major outer membrane proteins and 12 minor proteins in control bacteria outer membranes. Outer membrane protein content is drastically modified after treatments. In some cases, except for the major proteins OmpA and LamB, other outer membrane proteins seem to totally disappear. LamB is more resistant to hyperbaric exposure when the pH of the media is acidic. This behaviour could be explained by a different conformation adopted by the LamB protein depending on the extracellular pH. This work allows us to define membrane proteins as a target of high hydrostatic pressure treatments. Knowledge of the behaviour of these bacterial membrane proteins subjected to pressure under different conditions (pH, temperature, a(w)...) could allow an increase in the efficiency of treatments.

Bacterial Proteins↗
PubMed1987

Promoter recognition by Escherichia coli RNA polymerase: effects of base substitutions in the -10 and -35 regions.

We have constructed the PRM promoter of phage lambda and eight variants, which represents intermediates in the conversion of this promoter to one that has complete homology to the consensus sequences in the -10 and -35 regions. The in vivo activity of these promoters was determined from the beta-galactosidase or galactokinase activities in cells harboring plasmids, in which the cloned promoters were driving the expression of these genes. Additionally, the kinetics of the interaction of Escherichia coli RNA polymerase with the same series of promoters was measured as a function of RNA polymerase concentration. This allowed the overall rate of functional or open complex formation to be dissected into the equilibrium constant for binding of the polymerase to form a closed promoter complex and the rate of subsequent isomerization to yield the open complex. The following conclusions can be drawn from the data presented: (1) The consensus sequence is optimal for promoter function both in vivo and in vitro. (2) Alterations of the -10 and -35 regions have similar effects on the kinetics of RNA polymerase binding in vitro; with one exception, the same holds for promoter activity in vivo. (3) The in vitro rate of RNA polymerase binding to a promoter is solely determined by the number of positions at which its -10 and -35 regions match the consensus promoter sequence. The functional importance of a match does not appear to be determined by the sequence conservation at the particular position. (4) The extent to which a particular base change affects the kinetic parameters depends on the sequence of the promoter into which it is introduced.

Bacteriophage lambda↗
PubMed2003

Modeling anhydrous and aqua copper(II) amino acid complexes: a new molecular mechanics force field parametrization based on quantum chemical studies and experimental crystal data.

This paper presents the vacuum structures of aquacopper(II) bis(amino acid) complexes with glycine, sarcosine, N,N-dimethylglycine, and N-tert-butyl-N-methylglycine estimated using the B3LYP method. The differences between the B3LYP vacuum structures and experimental crystal structures suggested considerable influence of crystal lattice packing effects on the changes in the complexes' geometries. A previously developed molecular mechanics force field for modeling anhydrous copper(II) amino acidates was reoptimized to simulate these changes and predict the properties of both trans and cis anhydrous and aqua copper(II) amino acid complexes. The modeling included experimental molecular and crystal structures of 13 anhydrous and 10 aqua copper(II) amino acidates with the same atom types (Cu(II), C, H, N, and O) but various copper(II) coordination polyhedron geometries, crystal symmetries, and intermolecular interactions. The empirical parameters of the selected potential energy functions were optimized on the B3LYP vacuum copper(II) coordination geometries of three anhydrous copper(II) amino acidates and on experimental crystalline internal coordinates and unit cell dimensions of six anhydrous and six aqua copper(II) amino acid complexes. The respective equilibrium structures were calculated in vacuo and in simulated crystalline environment. The efficacy of the final force field, FFW, was examined. The total root-mean-square deviations between the experimental and theoretical crystal values were 0.018 A in the bond lengths, 2.2 degrees in the valence angles, 5.5 degrees in the torsion angles, and 0.395 A in the unit cell lengths. FFW reproduced the unit cell volumes in the range from -8.1 to 9.6%. The means of Cu to axial water oxygen distances were 2.4 +/- 0.1 A (experiment) and 2.6 +/- 0.1 A (FFW). This paper describes the ability of the molecular mechanics model and FFW force field to simulate the flexibility of the metal coordination polyhedron. The new force field proved effective in predicting the most stable molecular conformation of copper(II) amino acidato systems in vacuo.

Journal Article↗
PubMed1991

Comparison of estimates of cardiac output by indicator dilution and freon 22 uptake during gas mixing in dogs.

STUDY OBJECTIVE: The aim was to measure cardiac output while rebreathing tidal volumes, by correction of soluble gas uptake for gaseous mixing. DESIGN: Simultaneous measurements of cardiac output by indocyanin green and freon 22 uptake during rebreathing were made. Mixing for a hypothetical gas of identical gaseous diffusivity to freon 22 was calculated by interpolation between concentrations of two insoluble gases, helium and sulphur hexafluoride. Mixing efficiency was estimated by the number of breaths for helium to become 99% equilibrated with lung gas (n99-He). EXPERIMENTAL MATERIAL: Five anaesthetised dogs rebreathed at intervals with 300 ml of test gas. MEASUREMENTS AND MAIN RESULTS: 63 comparisons of cardiac output using indocyanin green and freon 22 uptake (over breaths 7-13 using the mean mixed volume of distribution), gave a mean (95% confidence interval) underestimation of 0.345 (0.093-0.597) litre.min-1 (14%). Exclusion of 12 points in which n99-He was greater than 15 resulted in a mean underestimation of 0.052(-0.163-0.267) litre.min-1 (2%). Without correction for gaseous mixing, freon 22 uptake for these data overestimated blood flow by a mean of 1.31 litre.min-1 (overestimation = 2.7 over breaths 5-11). Use of the equilibrium volume of distribution resulted in an overestimation of blood flow relative to green dye of 1.2 litre.min-1 (breaths 5-11) and 0.76 litre.min-1 (breaths 7-13). CONCLUSIONS: Estimates of cardiac output by soluble gas uptake are optimal when correction is made for mixing of gas of identical diffusivity. The mean mixed gas volume gives the best correlation with the reference method, implying a selective distribution of blood flow to the better ventilated areas.

Animals↗
PubMed2001

Two-time Green's functions and the spectral density method in nonextensive classical statistical mechanics.

The two-time retarded and advanced Green's function technique is formulated in nonextensive classical statistical mechanics within the optimal Lagrange multiplier framework. The main spectral properties are presented and a spectral decomposition for the spectral density is obtained. Finally, the nonextensive version of the spectral density method is given and its effectiveness is tested by exploring the equilibrium properties of a classical ferromagnetic spin chain.

Journal Article↗
PubMed2003

Role of ectoine in Vibrio cholerae osmoadaptation.

Vibrio cholerae is both an intestinal pathogen and a microbe in the estuarine community. To persist in the estuarine environment, V. cholerae must adjust to changes in ionic composition and osmolarity. These changes in the aquatic environment have been correlated with cholera epidemics. In this work, we study the response of V. cholerae to increases in environmental osmolarity. Optimal growth of V. cholerae in minimal medium requires supplementation with 200 mM NaCl and KCl. However, when the NaCl concentration is increased beyond 200 mM, a proportionate delay in growth is observed. During this delay in growth, osmotic equilibrium is reached by cytoplasmic accumulation of small, uncharged solutes that are compatible with growth. We show that synthesis of the compatible solute ectoine and transport of the compatible solute glycine betaine impact the length of the osmoadaptive growth delay. We also demonstrate that high-osmolarity-adapted V. cholerae displays a growth advantage when competed against unadapted cells in high-osmolarity medium. In contrast, low-osmolarity-adapted V. cholerae displays no growth advantage when competed against high-osmolarity-adapted cells in low-osmolarity medium. These results may have implications for V. cholerae population dynamics when seawater and freshwater and their attendant microbes mix.

Acetyltransferases↗
PubMed1998

Thermodynamics of propylparaben/beta-cyclodextrin inclusion complexes.

The aim of this study was to develop models for rigorous analysis of phase solubility diagrams, particularly the descending portion, in order to obtain individual thermodynamic complex formation and solubility product constants. Additionally, the effect of varying the initial solute concentration St, in excess of the optimal solubility Sm, on the general shape of the plateau and descending portions of the solubility diagram was investigated. The solubilities of propylpapraben (Seq) were measured against initial beta-cyclodextrin concentrations (Lt) at different temperatures and different St. Equilibrium concentrations (Leq) were also measured. The only effect observed was a broadening of the plateau region with increase in St with no discernible effect on Sm. Simultaneous rigorous analysis of the rising as well as the descending portions of the phase diagram could only be interpreted in terms of formation of two soluble complexes: SL and S2L. Rigorous analysis of the descending portion allowed the determination of individual formation constants K11 (SL) and K21 (S2L) in addition to the solubility product of the less soluble complex KS11 (SL). Thermodynamic analysis of the individual solubility of propylparaben and beta-cyclodextrin was carried out in water at different temperatures. In aqueous beta-cyclodextrin solutions, however, the solubility of propylparaben is enhanced due to the formation of both SL (delta G11(0) = -26.4 kJ/mol) and S2L (delta G21(0) = -46.4 kJ/mol) soluble complexes. Formation of the SL-complex is favored both by enthalpy (delta H11(0) = -20.7 kJ/mol) and a slight increase in entropy (delta S11(0) = 18.6 J/mol.K). Formation of S2L from SL apparently involves stronger solute-SL binding (delta H21(0) = -45.9 kJ/mol) yet is retarded by a net decrease in entropy (delta S21(0) = -86.3 J/mol.K). The solubility of the SL-complex is highly endothermic (delta HS11(0) = 55.8 kJ/mol), and although is accompanied by much randomization (delta SS11(0) = 101.8 J/mol.K), its solubility remains quite low (delta GS11(0) = 25.5 kJ/mol). Molecular mechanical modeling of propylparaben/beta-cyclodextrin interactions in water revealed that the S2L-complex formation is energetically more favored.

Cyclodextrins↗
PubMed1998

[A study on the binding of cyclodextrins with terbutaline enantiomers by chiral capilary zone electrophoresis].

The recognition mechanism of enantioselectivity in cyclodextrin-modified capillary zone electrophoresis (CD-CZE) on a racemic basic drug terbutaline was discussed. The equilibrium constants of host-guest complexation of terbutaline enantiomers with alpha-cyclodextrin, beta-cyclodextrin, 2,6-dimethyl-beta-cycoldextrin, 2,3,6-trimethyl-beta-cyclodextrin and 2-hydroxy-propyl-beta-cyclodextrin, and the thermodynamic parameters for chiral recognition procedure were determined respectively. It was found that the matching properties between cavity of cyclodextrins and enantiomers were interrelated with the equilibrium constants of binding complexes and selectivities(a) calculated from the ratio of binding constants of two enantiomers were in the same order of maximum electrophoretic mobility difference between two enantiomers responding with cyclodextrin additive. The experimental value of optimal concentration of cyclodextrin agreed with that calculated from the equation Copt = 1/(K1K2)1/2. From the thermodynamic parameter determination it was shown that the hydrogen-bonding interactions between terbutaline enantiomers and 2-hydroxy-propyl-beta-cyclodextrin and beta-cyclodextrin should be positive factors.

Cyclodextrins↗
PubMed1983

Fluorescence polarization immunoassay of estradiol.

Fluorescence polarization immunoassay (FPIA) offers a good alternative to isotopic methods for the determination of drug and hormone levels in biological fluids. We applied it to estradiol, using a fluoresceinated derivative of estradiol and the IgG fraction of a highly specific rabbit antiserum. After determining the optimal operational conditions, FPIA was used for measuring urinary concentrations of free estradiol in normal and pregnant women, after a purification step on a reversed phase cartridge. The method turned out to be rapid (1 min to reach the equilibrium state) and accurate.

Animals↗