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PubMed1979

Protein-protein interactions studied by counter-current distribution. I. Theoretical computations.

Many biological macromolecules are known to interact either with themselves, with other macromolecules or with small compounds. A simple equilibrium method for detecting and quantifying these interactions is to study the mutual influence of the molecules on their respective counter-current distribution in liquid-liquid biphasic systems. The theoretical counter-current distribution patterns for the components in an interacting system, A + B in equilibrium AB, have been calculated for two models in order to establish the boundary conditions and to optimize the experimental procedure. The patterns have been calculated for a range of association constants, partition coefficients and initial concentrations of the two reactants.

Countercurrent Distribution↗

PubMed2003

A chemometric approach to detection and characterization of multiple protein conformers in solution using electrospray ionization mass spectrometry.

Protein ion charge state distributions in electrospray ionization mass spectra have a potential to provide a wealth of information on protein dynamics, because they contain contributions from all protein conformers present in solution. Such ionic contributions often overlap, limiting the amount of useful information that can be extracted from the spectra. This difficulty is overcome in the present work by using a chemometric approach, which allows spectral deconvolution to be carried out and information on individual protein conformers to be extracted. Experiments are carried out by acquiring a series of spectra over a range of near-native and denaturing conditions to ensure significant changes in the conformers' populations. A total number of protein conformers sampled in the course of the experiments is determined by subjecting the set of collected spectra to singular value decomposition. Ionic contributions of each conformer to the total signal are then determined using a supervised optimization routine. Validation of the method has been carried out by monitoring acid- and alcohol-induced equilibrium states of well-characterized model proteins, chymotrypsin inhibitor 2 (two states), ubiquitin (three states) and apo-myoglobin (four states). For each of the model proteins, a new chemometric procedure yielded a picture of protein dynamics that was in excellent agreement with their documented behavior (as studied with other biophysical tools). The new method appears to be well-suited for monitoring protein dynamics in highly heterogeneous systems consisting of multiple proteins sampling a range of conformational states.

Algorithms↗

PubMed2004

Evaluation and optimization of an on-line admicelle-based extraction-liquid chromatography approach for the analysis of ionic organic compounds.

Admicelles-based solid-phase extraction (SPE) was on-line coupled with liquid chromatography/electrospray ionization/ion trap mass spectrometry, and it was proposed for the extraction of ionic organic compounds based on the formation of surfactant-analyte ion pairs. The approach was illustrated by studying the preconcentration of quaternary ammonium herbicides (quats) on sodium dodecyl sulfate (SDS) admicelles produced on alumina. Optimization of the parameters affecting SPE were studied on the basis that admicelles are dynamic entities in equilibrium with the aqueous phase. Some general guidelines could be established for method development from the results obtained. Factors influencing on-line operation were elucidated. On-line regeneration of the sorbent in each run was easily achieved by disruption of SDS admicelles with methanol and posterior coating of the alumina with SDS. The recovery of quats from drinking water samples were found quantitative for paraquat, diquat, and difenzoquat and above to 70% for chlormequat and mepiquat. Concentration factors of about 250, using sample volumes of 50 mL, were achieved. The detection limits ranged from 10 to 30 ng/L. The approach developed permits compliance with the directives of the European Community for drinking waters (100 ng/L) and goes deeply into the basis of solid-phase extractions that use surfactant-coated mineral oxide as sorbents.

Journal Article↗

PubMed1997

Engineering the independent folding of the subtilisin BPN' prodomain: analysis of two-state folding versus protein stability.

In complex with subtilisin BPN', the 77 amino acid prodomain folds into a stable compact structure comprising a four-stranded antiparallel beta-sheet and two three-turn alpha-helices. When isolated from subtilisin, the prodomain is 97% unfolded even under optimal folding conditions. Traditionally, to study stable proteins, denaturing cosolvents or temperatures are used to shift the equilibrium from folded to unfolded. Here we manipulate the folding equilibrium of the unstable prodomain by introducing stabilizing mutations generated by design. By sequentially introducing three stabilizing mutations into the prodomain we are able to shift the equilibrium for independent folding from 97% unfolded to 65% folded. Spectroscopic and thermodynamic analysis of the folding reaction was carried out to assess the effect of stability on two-state behavior and the denatured state. The denatured states of single and combination mutants are not discernably different in spite of a range of DeltaGunfolding from -2.1 to 0.4 kcal/mol. Conclusions about the nature of the denatured state of the prodomain are based on CD spectral data and calorimetric data. Two state folding is observed for a combination mutant of marginal stability (DeltaG = 0). Evidence for its two-state folding is based on the observed additivity of individual mutations to the overall DeltaGunfolding and the conformity of DeltaGunfolding vs T to two-state assumptions as embodied in the Gibbs-Helmholz equation. We believe our success in stabilizing the two-state folding reaction of the prodomain originates from the selection of mutations with improved ability to fold subtilisin rather than selection for increase in secondary structure content. The fact that a small number of mutations can stabilize the independent folding of the prodomain implies that most of the folding information already exists in the wild-type amino acid sequence in spite of the fact that the unfolded state predominates.

Bacillus subtilis↗

PubMed1999

The cleavage step of ribonuclease P catalysis is determined by ribozyme-substrate interactions both distal and proximal to the cleavage site.

The cleavage step of bacterial RNase P catalysis involves concentration-independent processes after the formation of the ribozyme-substrate complex that result in the breaking of a phosphodiester bond. The 2'OH group at the cleavage site of a pre-tRNA substrate is an important determinant in the cleavage step. We determined here that in contrast to a tRNA substrate, the 2'OH at the cleavage site of two in vitro selected substrates has no effect, whereas a 2'OH located adjacent to the cleavage site has a similarly large effect on the cleavage step. This result indicates that a unique 2'OH in the vicinity of the cleavage site interacts with the ribozyme to achieve the maximal efficiency of the cleavage step. Individual modifications in a pre-tRNA substrate that disrupt ES interactions proximal to the cleavage site generally have little effect on the usage of this unique 2'OH. Ribozyme modifications that delete the interactions involving the T stem-loop of the tRNA have a large effect on the usage of this unique 2'OH and also alter the location of this 2'OH. We propose a new ES complex prior to the bond-breaking step in the reaction scheme to explain these results. This second ES complex is in fast equilibrium with the initial ES complex formed by bimolecular collision. The ribozyme interaction with this unique 2'OH shifts the equilibrium in favor of the second ES complex. The formation of the second ES complex may require optimal geometry of the two independently folding domains of this ribozyme to precisely position crucial functional groups and Mg2+ ions in the active site. Such a domain geometry is significantly favored by the RNase P protein. In the absence of the protein, spatial rearrangement of these domains in the ES complex may be necessary.

Bacillus subtilis↗

PubMedDate not supplied

Combining synthetic and analytical strategies for preparative HPLC enantioseparation of monastrol racemic mixture.

Large-scale resolution of racemic monastrol has been carried out by normal-phase mode HPLC on an amylose-based chiral stationary phase. Because monastrol solubility, in media of proper compositions for normal-mode HPLC separations (in terms of retention factors and selectivity), was significantly low and impractical for preparative scopes, racemic monastrol was transformed into the corresponding O-tert-butyldimethylsilyl derivative. The tert-butyldimethylsilyl group was chosen as a suitable derivatizing agent because it induced approximatively a six-times higher solubility and allowed for an almost quantitative recovery of pure monastrol from the derivatization-deprotection sequence. The competitive isotherms of the O-tert-butyldimethylsilyl compounds, measured through frontal analysis, were fitted to competitive Langmuir and four-parameter bi-Langmuir models. The equilibrium dispersive model of chromatography was used for modeling the nonlinear separation of the racemate and to optimize the experimental conditions for collection of highly concentrated fractions of pure (R,S)-O-tert-butyldimethylsilyl compounds, from which significant amounts of the corresponding enantiomers of monastrol (about 100 mg of each enantiomer with 30 runs on an analytical-scale column) were obtained by quantitative back-derivatization.

Algorithms↗

PubMed2002

Coordination of the neptunyl ion with carbonate ions and water: a theoretical study.

The results of a study on the ground-state of monocarbonate, bicarbonate, and tricarbonate complexes of neptunyl using multiconfigurational second-order perturbation theory (CASSCF/CASPT2) are presented. The equilibrium geometries of the complexes corresponding to neptunium in the formal oxidation state (V) have been fully optimized at the CASPT2 level of theory in the presence of an aqueous environment modeled by a reaction field Hamiltonian with a spherical cavity. Some water molecules have been explicitly included in the calculation. This study is consistent with the hypothesis that the monocarbonate complex has a pentacoordinated structure with three water molecules in the first coordination shell and that the bicarbonate complex has a hexacoordinated structure, with two water molecules in the first coordination shell. The typical bond distances are in good agreement with experimental results. The tricarbonate complex was studied with explicit counterions, which resulted in somewhat longer Np-carbonate bond distances than experiment indicates.

Journal Article↗

PubMed2005

An ab initio study on thermal rearrangement reactions of 1-silylprop-2-en-1-ol H3SiCH(OH)CH=CH2.

The thermal rearrangement reactions of 1-silylprop-2-en-1-ol H3SiCH(OH)CH=CH2 were studied by ab initio calculations at the G2(MP2) and G3 levels. The reaction mechanisms were revealed through ab initio molecular orbital theory. On the basis of the MP2(full)/6-31G(d) optimized geometries, harmonic vibrational frequencies of various stationary points were calculated. The reaction paths were investigated and confirmed by intrinsic reaction coordinate (IRC) calculations. The results show that the thermal rearrangements of H3SiCH(OH)CH=CH2 happen in two ways. One is via the Brook rearrangement reactions (reaction A), and the silyl group migrates from carbon atom to oxygen atom passing through a double three-membered ring transition state, forming allyloxysilane CH2=CHCH2OSiH3. In the other, the reactant undergoes a dyotropic rearrangement; the hydroxyl group migrates from carbon atom to silicon atom coupled with a simultaneous migration of a hydrogen atom from silicon atom to carbon atom, forming allylsilanol CH2=CHCH2SiH2OH (reaction B). The barriers for reactions A and B were computed to be 343.5 and 203.7 kJ/mol, respectively, at the G3 level. The changes of the thermodynamic functions, entropy (DeltaS), entropy (DeltaS(doubledagger)) for the transition state, enthalpy (DeltaH), and free energy (DeltaG) were calculated by using the MP2(full)/6-31G(d) optimized geometries, and harmonic vibrational frequencies of reactants, transition states, and products with statistical mechanical methods, and equilibrium constant K(T) and reaction rate constant k(T) in canonical variational transition-state theory (CVT) with centrifugal-dominant small-curvature tunneling approximation (SCT) were calculated over a temperature range 400-1300 K. The conventional transition-state theory (TST) rate constants were also calculated for the purposes of comparison. The influences of the vinyl group attached to the center carbon of the alpha-silyl alcohols on reactions were discussed.

Journal Article↗

PubMed2003

Binding studies of the enzyme (factor IXa) with the cofactor (factor VIIIa) in the assembly of factor-X activating complex on the activated platelet surface.

Activated platelet membranes expose binding sites for the enzyme factor (F)IXa, the substrate (FX) and the cofactor (FVIIIa) that colocalize to assemble the FX-activating complex and promote optimal rates of FX activation. To determine the stoichiometry and affinity of binding to activated platelets, coordinate, equilibrium binding studies with enzyme (125I-FIXa) and cofactor (131I-FVIII or 131I-FVIIIa) were carried out in the presence of saturating concentrations of substrate (FX). Results of these studies indicate that in the presence of FX (1.5 micro m), the enzyme (active-site-inhibited Glu-Gly-Arg-FIXa, EGR-FIXa) and procofactor (FVIII) bind to an equal number (approximately 700 sites/platelet) of receptors whereas the active cofactor (FVIIIa) binds an additional approximately 500 high-affinity FVIIIa binding sites per platelet (Kd approximately 0.8 nm). With excess zymogen (FIX) to block shared FIX/FIXa-binding sites, the stoichiometry of 125I-FIXa and 131I-FVIIIa binding was 1:4. These FIXa/FVIIIa binding studies together with previously reported evidence of the coordinate binding of FVIIIa and FX to equivalent numbers of binding sites on activated platelets provide strong evidence to support the conclusion that FVIIIa comprises the receptor that presents FX to FIXa for efficient catalysis on the activated platelet membrane.

Autoradiography↗

PubMed2006

Basis-set extrapolation techniques for the accurate calculation of molecular equilibrium geometries using coupled-cluster theory.

To reduce remaining basis-set errors in the determination of molecular equilibrium geometries, a basis-set extrapolation (BSE) scheme is suggested for the forces used in geometry optimizations. The proposed BSE scheme is based on separating the Hartree-Fock and electron-correlation contributions and uses expressions obtained by straightforward differentiation of well established extrapolation formulas for energies when using basis sets from Dunning's hierarchy of correlation-consistent basis sets. Comparison with reference data obtained at the R12 coupled-cluster level [CCSD(T)-R12] demonstrates that BSE significantly accelerates the convergence to the basis-set limit, thus leading to improvements comparable to or even better than those obtained by increasing the cardinal number in the used basis set by one. However, BSE alone is insufficient to improve agreement with experiment, even after additional consideration of inner-shell correlation and quadruple-excitation effects (mean error and standard deviation with extrapolation are -0.014 and 0.047 pm in comparison with mean error and standard deviation of -0.002 and 0.036 pm without extrapolation). Improvement is obtained only when other contributions of similar magnitude as the BSE contributions (e.g., pentuple-excitation effects and relativistic effects) are also considered. A rather large discrepancy (of the order of a few tenths of a picometer) is observed for the F(2) molecule indicating an enhanced basis-set requirement for the various contributions in this case.

Journal Article↗

PubMed2006

Optimization of the anisotropic united atoms intermolecular potential for n-alkanes: improvement of transport properties.

The parameters of the anisotropic united atom (AUA) intermolecular potential for n-alkanes originally proposed by Toxvaerd [J. Chem. Phys. 93, 4290 (1990)] [AUA(3)] was optimized by Ungerer et al. [J. Chem. Phys. 112, 5499 (2000)] [AUA(4)] on the basis of equilibrium properties (vapor pressures, vaporization enthalpies, and liquid densities). In this work we analyze the influence of the torsion potential in the internal and collective dynamics of the AUA model. The modified potential [AUA(4m)] preserves all the intermolecular parameters and only explores an increment in the trans-gauche and gauche(+)-gauche(-) transition barrier of the torsion potential. This modification better reproduce different transport properties (shear viscosity, self-diffusion coefficient, and internal relaxation times), keeping the accuracy achieved in our previous work for equilibrium properties. An extensive investigation of the shear viscosity of ethane, n-pentane, n-dodecane, and n-eicosane in a wide range of pressures and temperatures shows that the AUA(4m) improves the accuracy of the original AUA(4), reducing the absolute average deviation from 30% to 14.5%. Finally, the self-diffusion coefficient of n-hexane computed with the new model in the range of 223-333 K and from 0.1 to 295 MPa is in better agreement with respect to the experimental data than the original model.

Journal Article↗

PubMed2001

An ab initio investigation of 2-amino-2-imidazoline: a key moiety in chemical and biochemical processes.

The 2-amino-2-imidazoline moiety is currently used not only in drugs, but also in insecticides, and fungicides. Ab initio calculations are performed to evaluate the molecular properties of the two tautomeric forms and the protonated form with extended basis sets ranging from 6-31G* to 6-311++G** at Hartree-Fock and density functional (BLYP and B3LYP) levels. Møller-Plesset perturbation is tested at the MP2/6-31G* level only. Optimized geometry structures, energies and thermochemical properties are generated. Basis set and correlation effects on geometries, tautomer equilibrium constant and protonation enthalpy are carefully analysed. Although observed for the isolated molecule, these results may be extrapolated to chemical and biochemical systems of interest.

Fungicides, Industrial↗

PubMedDate not supplied

Duplex polymerase chain reaction with confronting two-pair primers (PCR-CTPP) for genotyping alcohol dehydrogenase beta subunit (ADH2) and aldehyde dehydrogenase 2 (ALDH2).

AIMS: Alcohol dehydrogenase beta subunit (ADH2) Arg47His and aldehyde dehydrogenase 2 (ALDH2) Glu487Lys were genotyped by a duplex polymerase chain reaction (PCR) with confronting two-pair primers (PCR-CTPP), which allows DNA amplification with one-tube PCR including eight primers, and subsequent electrophoresis. METHODS: Several PCR conditions were tested to establish the optimal conditions for distinguishing the allele-specific bands for the two polymorphisms. Under the optimal PCR conditions, 454 Japanese health check-up examinees were genotyped. RESULTS: The allele-specific bands were successfully amplified under the optimal conditions of the duplex PCR-CTPP. The genotype distributions were within the Hardy-Weinberg equilibrium. The bands produced by the duplex PCR-CTPP genotyping were clearer than those produced by PCR-CTPP, conducted solely for ADH2. CONCLUSIONS: ADH2 Arg47His and ALDH2 Glu487Lys were successfully genotyped by this newly developed duplex PCR-CTPP, an inexpensive and time-saving genotyping tool, which will be useful in epidemiological studies on alcoholism, as well as risk estimation of alcohol-related diseases.

Adult↗

PubMed1982

Studies on the binding of a 32K rat epididymal protein to rat epididymal spermatozoa.

A glycoprotein of molecular weight 32K has been isolated and purified from the rat caudal epididymal fluid by gel filtration, ion-exchange and affinity chromatography. The highly purified protein was labeled with radioactive iodine and the binding of the 125I-labeled 32K rat epididymal protein (REP) to washed rat caudal epididymal sperm was studied under various conditions. Scatchard plots of the binding data revealed two binding kinetics. One bound with high affinity (KD = 2.6 X 10(-10) ) but low capacity. The other bound with lower affinity (KD = 2.2 X 10(-9)M) but high capacity. The rate of binding of the labeled protein to sperm was dependent on the temperature of the incubation medium. At the scrotal temperature of 33 degrees C, maximal binding was obtained after 40 min. However, at 22 degrees C equilibrium state was reached after 90 min and at 0 degrees C, the equilibrium rate was not reached even after 120 min of incubation. Binding showed dependence on extracellular pH (optimal pH at 4) and ionic strength of the incubation medium. High ionic strength was found to inhibit binding of the 125I-labeled 32K REP to rat caudal epididymal sperm. Specific binding was abolished by 100-fold molar excess unlabeled 32K REP or by native rat caudal epididymal fluid proteins, but not by albumin or ovalbumin. This indicates high specificity of binding. This study has provided direct evidence for the interaction of an epididymal protein with epididymal spermatozoa.

Animals↗

PubMed1981

Responses of Avena coleoptiles to suboptimal fusicoccin: kinetics and comparisons with indoleacetic Acid.

Proton excretion induced by optimal concentrations of indoleacetic acid (IAA) and fusicoccin (FC) differs not only in maximum rate of acidification but also in the lag before onset of H(+) excretion and in sensitivity to cycloheximide. Because these differences might simply be a consequence of the difference in rate of proton excretion, FC and IAA have now been compared using oat coleoptiles (cv. Victory) under conditions where the rates of acidification are more similar, i.e. suboptimal FC versus optimal IAA. As the concentration of FC is reduced, the rate of H(+) excretion decreases, the final equilibrium pH increases, and the lag before detectable acidification increases up to 7-fold. This enhanced lag period is not primarily a consequence of wall buffering, inasmuch as it persists when a low concentration of FC is added to sections which were already excreting H(+) in response to IAA. An extended lag also occurs, upon reduction of FC levels, in the hyperpolarization of the membrane potential, before enhancement of O(2) uptake and before the increased rate of Rb(+) uptake. The presence or absence of a lag is not a distinguishing feature between FC and IAA actions on H(+) excretion and cannot be used to discriminate between their sites of action. In contrast, the insensitivity of FC-induced H(+) excretion to cycloheximide, as compared with the nearly complete inhibition of this auxin effect by cycloheximide, persists even at dilute concentrations of FC. This seems to be a basic difference in H(+) excretion by IAA and FC.

Journal Article↗

PubMed1977

The denaturation-renaturation of chicken-muscle triosephosphate isomerase in guanidinium chloride.

1. The process of denaturation of the chicken muscle dimeric enzyme triosephosphate isomerase on addition of guanidinium chloride has been studied at pH 7.6, the pH at which the recovery of activity is optimal (100%) on removal of denaturant. Determinations of the sedimentation coefficient, intrinsic viscosity, molecular weight (by sedimentation equilibrium studies) and the absorption coefficient at 280 nm in various concentrations of guanidinium chloride concurred in showing a single, sharp transition at about 0.7 M guanidinium chloride at a protein concentration 1-5 mg/ml from the native enzyme to the dissociated, unfolded chains of the monomer. Relative fluorescent intensity measurements revealed a single transition at about 0.4 M guanidinium chloride at enzyme concentrations of about 0.05 mg/ml. 2. The process of denaturation in different guanidinium chloride concentrations was first order with respect to enzyme and about sixth order with respect to denaturant. 3. The rate of attainment of equilibrium during the renaturation obeyed second-order/first-order reversible kinetics. It was concluded that the rate-determining step in renaturation at pH 7.6 must be the association of two subunits.

Animals↗

PubMed1996

Antibodies to bovine serum albumin in Brazilian children and young adults with IDDM.

OBJECTIVE: To evaluate the prevalence of IgG antibodies to bovine serum albumin (BSA) in a cohort of Brazilian children and young adults with IDDM. RESEARCH DESIGN AND METHODS: Sera from 81 subjects with < 1 year of IDDM (group 1), III subjects with > 1 year of IDDM (group 2), and 207 normoglycemic subjects were tested using an immunofluorimetric assay. A receiver-operating-characteristic curve was used to establish the threshold of anti-BSA antibody titers defining the positivity of the assay. RESULTS: The distribution of the fluorimetric index (FI) of anti-BSA antibodies did not have a gaussian profile. Rank sum of FI was significantly higher in patients than in control subjects (P < 0.0001). Average logFI values of both IDDM groups were significantly higher than that of the control group (P < 0.005 for both groups). There was a trend toward higher FI levels in group 1 than in group 2 (P = 0.06). A FI cutoff of 0.7 optimized the ratio of true-positive to false-positive of the assay, with the best equilibrium between sensitivity and specificity. The prevalence of anti-BSA antibodies was 52% in group 1, 47% in group 2, and 28% in the control group (P = 0.0001). An independent association between anti-BSA antibodies and IDDM, with an odds ratio of 3.03 (P < 0.0001), was observed in a logistic regression analysis. However anti-BSA antibodies explained only 5% of the variability of IDDM versus NIDDM. CONCLUSIONS: Our results confirm that the prevalence of anti-BSA antibodies is higher in IDDM subjects than in control subjects, even after 1 year of diabetes. However, a large overlap of antibody titers is observed in patients and control subjects, suggesting that anti-BSA antibodies are neither sensitive nor specific markers of IDDM.

Adolescent↗

PubMed1999

Adsorptive control of water in esterification with immobilized enzymes. Continuous operation in a periodic counter-current reactor.

A periodic counter-current adsorptive-reactor system is developed to carry out continuous esterifications in organic solvents with immobilized enzymes. The system comprises a number of fixed-beds distributed between a reaction-adsorption zone and a regeneration zone and operated in a "merry-go-round" sequence. Water formed in the reaction is adsorbed preventing the formation of a free-water phase and deactivation of the biocatalyst. The adsorbed water is, in turn, recovered by desorption in the regeneration zone. The concept is tested experimentally on a laboratory-scale using, as a model, the esterification of isoamyl alcohol and propionic acid in hexane catalyzed by an immobilized lipase. Pure isoamyl alcohol is used as a regenerant to remove excess water from the biocatalyst. In the periodic steady-state, improvements in ester productivity greater than 50% over that achievable with a conventional fixed-bed reactor are demonstrated experimentally with just two beds in a series arrangement. Use of a water-selective adsorbent in conjunction with the biocatalyst provides further improvements by reducing accumulation of water on the enzyme. A mathematical model is also developed to predict the thermodynamic activity of water along the reactor and describe the dynamic behavior of the system. The model, based on independently developed rate and equilibrium parameters, successfully predicts the experimental behavior and provides an effective tool for scale-up and optimization.

Adsorption↗