Skeletal structure of clathrin triskelion in solution: experimental and theoretical approaches

The physicochemical properties of the clathrin triskelion were determined by dynamic and static light-scattering and sedimentation analyses in Tris and triethanolamine (TEA) buffers of about pH 8, in which the clathrin triskelion has been found to be in different conformational states by electron microscopy [Heuser, J., & Kirchhausen, T. (1985) J. Ultrastruct. Res. 92, 1-27]. Dynamic light-scattering measurements provided diffusion coefficients (D0(20,w)) of 1.22 x 10(-7) and 1.23 x 10(-7) cm2/s, and ultracentrifugal analysis gave sedimentation coefficients (S0(20,w)) of 8.39 and 8.32 S in Tris and TEA buffer, respectively. The average Stokes radius of the protein was determined to be 175 A from its diffusion and sedimentation coefficients and its molecular weight. Static light-scattering analysis provided molecular weights of 6.58 x 10(5) and 6.41 x 10(5) and radii of gyration of 311 and 301 A in the respective buffers. These results indicate that the clathrin triskelion has a similar conformation in the two buffers. For clarification of the skeletal structure of the clathrin triskelion in solution, the physicochemical parameters were calculated by using two models in which the clathrin arms are bent at various angles in a plane, on the basis of the Bloomfield approximation and a formula derived to estimate the radius of gyration of proteins consisting of various structural units. Values for the Stokes radius, diffusion and sedimentation coefficients, and radius of gyration in the ranges of 178-170 A, (1.20-1.26) x 10(-7) cm2/s, 8.26-8.66 S, and 316-266 A, respectively, were obtained with these models with the arms bent in the range of 0-60 degrees.(ABSTRACT TRUNCATED AT 250 WORDS)     

Taurocholate- and taurochenodeoxycholate-lecithin micelles: The equilibrium of bile salt between aqueous phase and micelle

The equilibrium of bile salt between aqueous phase and mixed micelle was studied in solutions of pure bile salt and lecithin comparing taurocholate and taurochenodeoxycholate. The relationship between bile salt concentration in the aqueous phase and the ratio of bile salt/lecithin in the mixed micelle was determined by equilibrium dialysis on serial dilutions of these solutions. Extrapolation of this relationship to zero mixed-micellar bile salt permitted calculation of the critical micelle concentration (CMC) of the mixed micelle. For taurocholate, taurochenodeoxycholate, and an equimolar mix of these two bile salts, the mixed micelle CMC's were 3.1 mM, 0.47 mM, and 0.89 mM respectively. In the most concentrated solutions, aqueous phase bile salt concentration surpassed the CMC of the simple bile salt micelle by more than four-fold indicating the presence of simple micelles as well as mixed micelles. At all dilutions taurochenodeoxycholate had a much greater affinity for the mixed micelle than did taurocholate. This last finding may be the reason for the superior cholesterol solubilizing capacity of taurochenodeoxycholate-lecithin solutions compared to taurocholate-lecithin solutions.     

Dynamic stereochemistry of rutin (vitamin P) in solution: theoretical approaches and experimental validation

Rutin, vitamin P, was extracted from Salvia macrosiphon and identified by ¹H, ¹³C, ¹H-¹H COSY, HMQC, and HMBC spectroscopy. In parallel, density functional theory (DFT) using B₃LYP functional and split-valance 6-311G∗∗ basis set has been used to optimize the structures and conformers of rutin. Also experimental and theoretical methods have been used to correlate the dependencies of ¹J, ²J, and ³J involving ¹H and ¹³C on the C5″-C6″ (ω), C6″-O6″ (θ), and C1?-O6″ (φ) torsion angles in the glycosidic moiety. New Karplus equations are proposed to assist in the structural interpretation of these couplings. ³J_HH depends mainly on the C-C (ω) torsion angle, as expected, and ²J_HH values depend on both C-C (ω) and C-O (θ) torsions. ¹J_CH values within hydroxymethyl fragments were also examined and found to depend on r_CH, which is modulated by specific bond orientation and stereoelectronic factors. In all calculations solvent effects were considered using a polarized continuum model (PCM).     

Raman spectroscopic analysis of the sodium salt of kappa-carrageenan and related compounds in solution

Laser-Raman spectra of Na⁺ kappa-carrageenan, Na⁺ neocarrabiose 4-sulphate, and neocarrabiose in the region 700–1500 cm⁻¹ are reported for solutions in H₂O and D₂O. The C-1-H-1α vibration, coupled with COH related modes, is assigned to a band at 840 cm⁻¹, close to the maximum of the symmetrical COS stretching (∼850 cm⁻¹). The symmetrical SO stretch is proposed to occur near 1040 cm⁻¹ and is probably coupled with COH vibrations which give rise to strong bands in the region 1000–1100 cm⁻¹. The intense band in the region 730–740 cm⁻¹ is ascribed to a complex ring vibration.     

Fusion pore conductance: experimental approaches and theoretical algorithms.

Time-resolved admittance measurements provide the basis for studies showing that membrane fusion occurs through the formation and widening of an initially small pore, linking two previously separated aqueous compartments. Here we introduce modifications to this method that correct the cell-pipette (source) admittance for attenuation and phase shifts produced by electrophysiological equipment. Two new approaches for setting the right phase angle are discussed. The first uses the displacement of a patch-clamp amplifier C-slow potentiometer for the calculation of phase. This calculation is based on amplitudes of observed and expected (theoretical) changes in the source admittance. The second approach automates the original phase adjustment, the validity of which we prove analytically for certain conditions. The multiple sine wave approach is modified to allow the calculation of target cell membrane parameters and the conductance of the fusion pore. We also show how this technique can be extended for measurements of the resting potential of the first (voltage-clamped) membrane. We introduce an algorithm for calculation of fusion pore conductance despite a concurrent change in the resistance of the clamped membrane. The sensitivity of the capacitance restoration algorithm to phase shift errors is analyzed, and experimental data are used to demonstrate the results of this analysis. Finally, we show how the phase offset can be corrected "off-line" by restoring the shape of the capacitance increment.     

Unblocked statistical-coil tetrapeptides and pentapeptides in aqueous solution: a theoretical study

NMR studies of the molecular conformations of peptides and proteins rely on a comparison of the relevant spectral parameters with the corresponding values for so-called statistical-coilpolypeptides. For this reason, it is necessary to characterize the experimental ensemble of states populated by statistical-coilpeptides. Such a characterization, however, has proven to be both difficult and sensitive to changes in many environmental parameters such as solvent composition, temperature, pH, as well as the neighboring amino acids in the sequence. As a consequence, a series of significant discrepancies has been reported for some experimentally observed parameters, such as chemical shifts, or vicinal coupling constants, ³J_NH, whose values appear to be incompatible with a statistical-coilensemble. In this work, we report the results of a molecular mechanics study of a series of unblocked tetra- and pentapeptides under different pH conditions. These calculations were carried out with explicit consideration of both the coupling between the process of proton binding/release and conformation adopted by the molecule at a given pH and the contribution of the conformational entropy to the total free energy. Good agreement was found between the calculated and experimentally determined values of the vicinal coupling constant, ³J_NH, the -proton chemical shift, and the ¹³Cchemical shift. All the evidence accumulated in these theoretical calculations helps to rationalize some of the unsettled anomalies observed experimentally, and to provide an understanding of the effect of pH and amino acid sequence on the conformational preferences of statistical-coilpeptides.     

Experimental and theoretical approaches for Cd(II) biosorption from aqueous solution using Oryza sativa biomass

Biomass of Oryza sativa (OS) was tested for the removal of Cd(II) ions from synthetic and real wastewater samples. Batch experiments were conducted to investigate the effects of operating parameters on Cd(II) biosorption. Fourier transform infrared spectroscopy, scanning electron microscopy, and energy-dispersive x-ray spectroscopy were used to examine the surface characteristics of the Cd(II)-loaded biomass. The maximum removal efficiency of Cd(II) was 89.4% at optimum pH 6.0, biosorbent dose 10.0 g L^?1, initial Cd(II) 50 mg L^?1, and biosorbent particle size 0.5 mm. The applicability of Langmuir and Freundlich isotherms to the sorbent system implied the existence of both monolayer and heterogeneous surface conditions. Kinetic studies revealed that the adsorption process of Cd(II) followed the pseudo-second-order model (r2: 0.99). On the theoretical side, an adaptive neuro-fuzzy inference system (ANFIS) was applied to select the operating parameter that mostly influences the Cd(II) biosorption process. Results from ANFIS indicated that pH was the most influential parameter affecting Cd(II) removal efficiency, indicating that the biomass of OS was strongly pH sensitive. Finally, the biomass was confirmed to adsorb Cd(II) from real wastewater samples with removal efficiency close to 100%. However, feasibility studies of such systems on a large-scale application remain to be investigated.     

A theoretical study of glucose mutarotation in aqueous solution

In this work the mechanism of glucose mutarotation is investigated in aqueous solution considering the most likely pathways proposed from experimental work. Two mechanisms are studied. The first involves an intramolecular proton transfer as proposed by textbooks of organic chemistry, and the second uses one solvent water molecule to assist proton transfer. Both mechanisms are studied in the gas phase and in aqueous solution with the help of a polarizable continuum model, which is adopted to introduce the electrostatic nonspecific influence of bulk solvent. The structures are fully characterized through the calculation of the corresponding vibrational frequencies. The rate coefficients for each mechanism are calculated following transition-state theory in both the gas phase and in aqueous solution. Values computed for the water-assisted pathway in the continuum solvent agree best with the experimental results.     

Effect of the cluster size on the micro phase separation in mixtures of beta-lactoglobulin clusters and kappa-carrageenan

The phase separation of globular protein clusters formed by heat-denatured beta-lactoglobulin (beta-lg) in mixtures with the polysaccharide kappa-carrageenan (kappa-car) has been studied at pH 7 and 20 degrees C. The effect of the protein cluster size on the phase separation was investigated by preparing clusters with radii between 20 nm and 1 mum. The formation of protein rich microdomains led to an increase of the turbidity starting at a minimum kappa-car concentration that decreased with increasing cluster size, but was only weakly dependent on the protein concentration. The size and number of microdomains do not depend much on the cluster size, but their density decreases with increasing cluster size leading to a lower turbidity.     

Genetic transformation of sugar beet: evolution of theoretical and experimental approaches

The review is dedicated to several aspects of sugar beet (Beta vulgaris L.) biotechnology: in vitro cultivation, callus induction, plant regeneration and genetic transformation. Media composition, methods of plant regeneration via somatic embryogenesis and protoplast culture are analysed. The use of Agrobacterium tumefaciens and gold particle bombardment is the base for modern genetic transformation methods.     

Natural and accelerated recovery from brain damage: experimental and theoretical approaches

The goal of the Caltech group is to gain insight into the processes that occur within the primate nervous system during dexterous reaching and grasping and to see whether natural recovery from local brain damage can be accelerated by artificial means. We will create computational models of the nervous system embodying this insight and explain a variety of clinically observed neurological deficits in human subjects using these models.     

The evaluation of Cr-curcumin-DNA complexation by experimental and theoretical approaches

Chromium(III) chloride mediates DNA-DNA cross-linking. Some chromium complexes promote programmed cell death in specific ligand environment through binding to DNA. One strategy that can be supposed for reduction of Cr³⁺ binding affinity to DNA is using curcumin as a chelator. In the current study, the [Cr(Curcumin)(EtOH)₂](NO₃)₂ (CCC) was synthesized and characterized by UV/Vis, FT-IR, CHN and spectrophotometric titration techniques. The mole ratio plot revealed a 1:1 complex between Cr³⁺ and curcumin in solution. Binding interaction of this complex with calf thymus-DNA (CT-DNA) was investigated using UV/Vis, circular dichroism (CD), FT-IR and cyclic voltammetry. The intrinsic binding constants of CCC with DNA, measured by UV/Vis and cyclic voltammetry, were 1.60 × 10⁵ and 1.13 × 10⁵, respectively. The thermodynamic studies showed that the reaction is enthalpy and entropy favoured. CD analysis revealed that only Λ-CCC interacts with DNA and Δ-CCC form has no tendency towards DNA. Based on FT-IR studies, it was understood that CCC interacts with DNA via minor groove binding. The docking simulation was carried out for finding the binding mode of CCC to DNA, too. All of data demonstrated that the curcumin significantly reduced the affinity of Cr³⁺ to the DNA and the form of Δ-CCC has no interaction with DNA.     

Interaction of bile salt monomer and cholesterol in the aqueous phase

The purpose of the present study was to evaluate the possible interaction of bile salt monomer and cholesterol in the intermicellar aqueous phase. Cholesterol and taurocholate monomer concentrations in the intermicellar aqueous phase were determined using 0-20 mM taurocholate solutions saturated with cholesterol. Maximal solubilities of cholesterol in aqueous solutions having various concentrations of taurocholate, especially below its intermicellar monomer concentration (critical micellar concentration), were determined and compared with the intermicellar cholesterol concentration. The intermicellar monomer concentration of taurocholate was constant (6 mM) and independent of taurocholate concentrations. The cholesterol concentration in the intermicellar aqueous phase gradually increased, depending upon taurocholate concentrations, and became constant (1,3 microM) above 10 mM taurocholate. The solubility of cholesterol increased linearly with the taurocholate concentration even below the critical micellar concentration, and was 0.3 microM at 6 mM taurocholate, which was approx. 20-times higher than the aqueous solubility of cholesterol, but a fifth of the maximal intermicellar cholesterol concentration. The results indicate that the higher cholesterol concentration in the intermicellar aqueous phase compared to its aqueous solubility can be primarily ascribed to the interaction of cholesterol with bile salt monomers possibly forming bile salt-cholesterol dimers, and partly to the sustaining forces induced by numerous micelles.     

Combining theoretical and experimental approaches to understand the circadian clock

This review is intended as a summary of our work carried out as part of the German Research Association (DFG) Center Program on Circadian Rhythms. Over the last six years, our approach to understanding circadian systems combined theoretical and experimental tools, and Gonyaulax and Neurospora have proven ideal for these efforts. Both of these model organisms demonstrate that even simple circadian systems can have multiple light input pathways and more than one rhythm generator. They have both been used to elaborate basic circadian features in conjunction with formal models. The models introduce the “zeitnehmer,” i.e., a clock-regulated input pathway, to the conceptual framework of circadian systems, and proposes networks of individual feedbacks as the basis for circadian rhythmicity.     

On the kinetics of phase separation in aqueous two-phase systems

The effect of the tie-line location (phase volume ratio) on the kinetics of phase separation in batch PEG/salt aqueous two-phase systems (ATPS) has been investigated. PEG/sulphate systems with a stability ratio (sr) of 0.34 and 0.37 and relative tie-line lengths in the range 0.1 to 0.6 for a continuous top phase and in the range 0.03 to 0.15 for a continuous bottom phase were used in the batch studies. A continuous settler was designed with three different inlet geometries. Phase separation is much faster when the bottom phase is continuous and in this case the location on the tie-line and the presence or absence of Bacillus subtilis extract makes little difference. When the top phase is continuous the relative sizes of the phases (phase ratio, R, relative distance on tie-line, rd) has an important effect, the larger the top phase (larger R and rd) the slower the phase separation. The presence of Bacillus extract also makes the operation slower which is more marked at the largest values of R (and rd). At the largest volume ratios (R or rd) three different settling regions have been recognised, a region of coalescence, a region of drops moving to the interphase and a region where drops queue at the interphase to coalesce into the large phase. A modified correlation that takes into account the location on the tie-line and thus volume ratio (R) and relative distance (rd) has been proposed and successfully tested. The behavior of batch and continuous systems in the presence and absence of Bacillus subtilis extract in systems with continuous bottom phase was also studied. The settling velocity was lower in the continuous than in the batch systems, and in both cases the initial rate was lower in the presence of Bacillus extract.     

Protein phase behavior in aqueous solutions: crystallization, liquid-liquid phase separation, gels, and aggregates

The aggregates and gels commonly observed during protein crystallization have generally been considered disordered phases without further characterization. Here their physical nature is addressed by investigating protein salting-out in ammonium sulfate and sodium chloride for six proteins (ovalbumin, ribonuclease A, soybean trypsin inhibitor, lysozyme, and β-lactoglobulin A and B) at 4°C, 23°C, and 37°C. When interpreted within the framework of a theoretical phase diagram obtained for colloidal particles displaying short-range attractive interactions, the results show that the formation of aggregates can be interpreted theoretically in terms of a gas-liquid phase separation for aggregates that are amorphous or gel-like. A notable additional feature is the existence of a second aggregation line observed for both ovalbumin and ribonuclease A in ammonium sulfate, interpreted theoretically as the spinodal. Further investigation of ovalbumin and lysozyme reveals that the formation of aggregates can be interpreted, in light of theoretical results from mode-coupling theory, as a kinetically trapped state or a gel phase that occurs through the intermediate of a gas-liquid phase separation. Despite the limitations of simple theoretical models of short-range attractive interactions, such as their inability to reproduce the effect of temperature, they provide a framework useful to describe the main features of protein phase behavior.     

Erythrocyte lysis in isotonic solution of ammonium chloride: theoretical modeling and experimental verification

A mathematical model of erythrocyte lysis in isotonic solution of ammonium chloride is presented in frames of a statistical approach. The model is used to evaluate several parameters of mature erythrocytes (volume, surface area, hemoglobin concentration, number of anionic exchangers on membrane, elasticity and critical tension of membrane) through their sphering and lysis measured by a scanning flow cytometer (SFC). SFC allows measuring the light-scattering pattern (indicatrix) of an individual cell over the angular range from 10° to 60°. Comparison of the experimentally measured and theoretically calculated light scattering patterns allows discrimination of spherical from non-spherical erythrocytes and evaluation of volume and hemoglobin concentration for individual spherical cells. Three different processes were applied for erythrocytes sphering: (1) colloid osmotic lysis in isotonic solution of ammonium chloride, (2) isovolumetric sphering in the presence of sodium dodecyl sulphate and albumin in neutrally buffered isotonic saline, and (3) osmotic fragility test in hypotonic media. For the hemolysis in ammonium chloride, the evolution of distributions of sphered erythrocytes on volume and hemoglobin content was monitored in real-time experiments. The analysis of experimental data was performed in the context of a statistical approach, taking into account that parameters of erythrocytes vary from cell to cell.     

Proton affinity of para-substituted acetophenones in gas phase and in solution: a theoretical study

The gas phase proton affinities PA and basicities GB for a series of para-substituted acetophenones weak bases (B) p.X???C₆H₄CO*CH₃ with X?=?H, F, Cl, Br, I, Me, CF₃, CN, NO₂, OCH₃, NH₂, CH₂OH, N(CH₃)₂, OH, $ {\text{NH}}_3^{+} $ , … have been calculated at 298.15 K at the density functional theory DFT/B3LYP level with a 6-311++G (2d,2p) basis set. Conformational results lead to only one stable planar conformer for both unprotonated compounds and their O*-protonated forms. Satisfactory accuracy and computational efficiency could be reached if the computed PAs are scaled by a factor 0.983. Protonation at more than one site is discussed and the carbonyl oxygen atom is found to be the preferential protonated site rather than the substituent X. The calculated gas phase PAs show a good agreement with the experimental available data. The electron-donating/electron-withdrawing nature of the substituents has an enormous influence upon the thermochemical and structural properties. The influence of environment on the proton affinity has been studied by means of SCRF solvent effect computations using PCM solvation model for two solvents: water and SO₂CI₂. Confrontation between computed and experimental pK(B) values exhibits better agreement in aqueous solution than in organic solvent.