“Ordered” structure in solutions and gels of a globular protein as studied by small angle neutron scattering

Small-angle neutron scattering measurements were used for structural investigation of β-lactoglobulin solutions and heat-set gels in conditions of strong double layer repulsions. At pH 9 and low ionic strength, a correlation peak was observed on the scattering curves of the solutions whatever the protein concentration C used (in the range C = 0.02–0.10 g/mL). The wave vector value q_max corresponding to these maxima scaled as C^0.25. This exponent value is in agreement with those reported in the literature for other globular proteins in the same concentration range. Increasing the ionic strength decreased the peak which vanished without changing position at 0.1M NaCl. This polyelectrolyte-like behaviour suggests a local structure in the protein solution due to double layer repulsions. In the case of heat-set aggregates and gels (0.02–0.13 g/mL) formed at pH 9 and low ionic strength, a peak in the scattering curves was also observed indicating that even after gelation a correlation is still present; q_max varied as C^0.5. As in the case of the solutions, the correlation peak decreased with increasing ionic strength, and it vanished at 0.06M NaCl. The dilution of the aggregates in order to determine their intraparticle structure factor showed that the correlations were lost and that the aggregates displayed the same internal structure as the elementary subunit in the gels. At high ionic strength, fractal structures of the aggregates down to a length scale of about 40 Å were observed with d_f = 1.3–1.75 ± 0.05, increasing with protein concentration. Subsequent dilution didn't change the fractal dimension of these structures. © 1996 John Wiley & Sons, Inc.     

Water transport in invertebrate peripheral nerve fibers

Osmotic and diffusion permeabilities (P_f and P_d) of invertebrate nerve fibers to tritiated water were measured to determine what water flux studies could reveal about "the nerve membrane" and to directly test the possibility of active transport of water into or out of invertebrate nerve fibers. P_f/P_d ratios for lobster walking leg nerve fibers were found to be about 20 ± 7 at 14°C. P_d measurements were made for squid giant axons at 25°C. and found to yield a value of 4 x 10^–4 cm.^–1 sec.^–1. When combined with the data of D. K. Hill for P_f, a P_f/P_d ratio of 21 ± 5 is obtained. These P_f/P_d ratios correspond to "effective pore radii" of about 16 ± 4 angstrom units, according to theories developed by Koefoed-Johnsen and Ussing and independently by Pappenheimer and his colleagues. Variations of water flux ratios with temperatures were studied and apparent activation energies calculated for both diffusion experiments and osmotic filtration experiments using the Arrhenius equation, and found to be close to 3 to 5 cal. per mole of water transferred. Cyanide (5 x 10^–3 molar) and iodoacetate (1 x 10^–3 molar) poisoned lobster leg nerve fibers showed no appreciable change in diffusion or osmotic filtration water effluxes. Caution in interpreting these proposed channels as simple pores was emphasized, but the possibility that such channels exist and are related to ionic flow is not incompatible with electrophysiological data.     

Influence of sodium concentration on changes of membrane capacitance associated with the electrogenic ion transport by the Na,K-ATPase

Electrogenic ion transport by the Na,K-ATPase was investigated in a model system of protein-containing membrane fragments adsorbed to a lipid bilayer. Transient Na⁺ currents were induced by photorelease of ATP from inactive caged ATP. This process was accompanied by a capacitance change of the membrane system. Two methods were applied to measure capacitances in the frequency range 1 to 6000 Hz. The frequency dependent capacitance increment, ΔC, was of sigmoidal shape and decreased at high frequencies. The midpoint frequency, f ₀, depended on the ionic strength of the buffer. At 150 mm NaCl f ₀ was about 200 Hz and decreased to 12 Hz at high ionic strength (1 M). At low frequencies (f≪f ₀) the capacitance increment became frequency independent. It was, however, dependent on Na⁺ concentration and on the membrane potential which was generated by the charge transferred. A simple model is presented to analyze the experimental data quantitatively as a function of two parameters, the capacitance of the adsorbed membrane fragments, C _P, and the potential of maximum capacitance increment, ψ ₀. Below 5 mm Na⁺ a negative capacitance change was detected which may be assigned to electrogenic Na⁺ binding to cytoplasmic sites. It could be shown that the results obtained by experiments with the presented alternating current method contain the information which is determined by current-relaxation experiments with cell membranes. Received: 3 November 1997 / Revised version: 19 February 1998 / Accepted: 21 February 1998     

G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA

It has been previously shown that the DHX36 gene product, G4R1/RHAU, tightly binds tetramolecular G4-DNA with high affinity and resolves these structures into single strands. Here, we test the ability of G4R1/RHAU to bind and unwind unimolecular G4-DNA. Gel mobility shift assays were used to measure the binding affinity of G4R1/RHAU for unimolecular G4-DNA-formed sequences from the Zic1 gene and the c-Myc promoter. Extremely tight binding produced apparent K_d’s of 6, 3 and 4 pM for two Zic1 G4-DNAs and a c-Myc G4-DNA, respectively. The low enzyme concentrations required for measuring these K_d’s limit the precision of their determination to upper boundary estimates. Similar tight binding was not observed in control non-G4 forming DNA sequences or in single-stranded DNA having guanine-rich runs capable of forming tetramolecular G4-DNA. Using a peptide nucleic acid (PNA) trap assay, we show that G4R1/RHAU catalyzes unwinding of unimolecular Zic1 G4-DNA into an unstructured state capable of hybridizing to a complementary PNA. Binding was independent of adenosine triphosphate (ATP), but the PNA trap assay showed that unwinding of G4-DNA was ATP dependent. Competition studies indicated that unimolecular Zic1 and c-Myc G4-DNA structures inhibit G4R1/RHAU-catalyzed resolution of tetramolecular G4-DNA. This report provides evidence that G4R1/RHAU tightly binds and unwinds unimolecular G4-DNA structures.     

Some comments on the conductance of slow inward current in cardiac muscle

Voltage clamp experiments, which determine the kinetic parameters of calcium conductance of cardiac muscle, (d _∞,f _∞, τ _d and τ _f ) are analyzed with a generally accepted expression for slow inward currentI _s=g _sdf (E-E _R). Activation (d) and inactivation (f) reach the final valuesd _∞ andf _∞ with time constants τ _d and τ _f respectively. The analysis indicates that the measuredf _∞ agrees with the theoreticalf _∞, but the measuredd _∞ differs from the theoreticald _∞ by a factor which depends on τ _d . The peak tension can be made to correlate closely with the theoreticald _∞ after a correction factor is applied to the raw measurements of activation. It can be shown that experiments designed to measure τ _f can also be used to determine τ _d with greater accuracy.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome c₁ and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c₁ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c₁ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c₁ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Theory and Simulation of Water Permeation in Aquaporin-1

We discuss the difference between osmotic permeability p_f and diffusion permeability p_d of single-file water channels and demonstrate that the p_f/p_d ratio corresponds to the number of effective steps a water molecule needs to take to permeate a channel. While p_d can be directly obtained from equilibrium molecular dynamics simulations, p_f can be best determined from simulations in which a chemical potential difference of water has been established on the two sides of the channel. In light of this, we suggest a method to induce in molecular dynamics simulations a hydrostatic pressure difference across the membrane, from which p_f can be measured. Simulations using this method are performed on aquaporin-1 channels in a lipid bilayer, resulting in a calculated p_f of 7.1 × 10⁻¹⁴ cm³/s, which is in close agreement with observation. Using a previously determined p_d value, we conclude that p_f/p_d for aquaporin-1 measures ~12. This number is explained in terms of channel architecture and conduction mechanism.     

Effect of aneurysm on biomechanical properties of “radially-oriented” collagen fibers in human ascending thoracic aortic media

We recently reported a mechanistic model to link micro-architectural information to the delamination strength (S_d) of human ascending thoracic aorta (ATA). That analysis demonstrated that the number density (N) and failure energy (U_f) of the radially-oriented collagen fibers contribute to the S_d of both aneurysmal (ATAA) and non-aneurysmal (CTRL-ATA) aortic tissue. Among the set of ATAA samples, we studied specimens from patients displaying bicuspid (BAV) and tricuspid aortic valve (TAV) morphologic phenotypes. Results from our prior work were based on the assumption that the U_f was independent of dissection direction. In the current study, we excluded that assumption and hypothesized that U_f correlates with the S_d of ATAA. To test the hypothesis, we used previously-reported experimentally-determined S_d measurements and N of radially-oriented collagen fibers as input in our validated mechanistic model to calculate U_f for BAV-ATAA, TAV-ATAA and CTRL-ATA tissue specimens. The results of our analysis revealed that U_f is significantly lower for both BAV-ATAA and TAV-ATAA compared to CTRL-ATA cases, and does not differ between BAV-ATAA and TAV-ATAA. Furthermore, we found that U_f is consistent between circumferential-radial and longitudinal-radial planes in either of BAV-ATAA, TAV-ATAA or CTRL-ATA specimens. These findings employ a novel mechanistic model to increase our understanding of the putative interrelationship between biomechanical properties, extracellular matrix biology, and failure energy of aortic dissection.     

Relation between mechanical foam-breaking difficulty and the foaming characteristics of solutions

Using a small foaming apparatus, liquid foaming characteristics such as liquid holdup in foamate flow (φ_t), rate of liquid drainage from foam (ν), foam velocity (u_f) and foam size (d_f) were examined for various liquids. A parameter φ_f, incorporating φ_t, ν and u_f, was obtained. The relationship among φ_f, d_f and the liquid holdup in foam, φ_b, in the actual gas-bubbling systems under foam control was expressed in the form of φ_b ∞ φ_fd_f^1.83, regardless of the type of foaming liquid or its concentration. This relationship was useful for the prediction of φ_b as related to mechanical foam-breaking difficulty.     

Characterization of the physical properties of yeast flocs

Summary Physical characteristics, namely floc density function, floc size distribution, and relative floc strength, of a number of flocculent yeast types were measured. A straight-line relationship was found to exist between log values of size and density for the yeasts examined. Each yeast type had coefficients from this relationship which could be used to interpret settling behaviour. Indices of relative floc strength were also obtained and together with the floc density function allowed fuller interpretation of yeast settling than with simpler theories.Symbols a constant (g·cm^-3) - B ₂/B ₁ floc binding strength of floc₂ relative to floc₁ - d _f floc diameter (cm) - d _i image diameter on print (cm) - d _max maximum floc diameter (cm) - f _d Ploc effective density (g·cm^-3) - g gravitational constant (981 cm·s^-1) - K _p constant (-) - R _l rate of enlargement on film - R ₂ rate of enlargement on print - S _s density of suspending liquid phase (g·cm^-3) - S _f density of solid (floc) phase (g·cm^-3) - U _t terminal settling velocity (cm·s^-1) - u liquid viscosity (g·cm^-3·^-1)     

Electrostatic effects on polynucleotide transitions. II. Behavior of titrated systems

The theory developed in the previous paper to discuss changes in electrostatic free energies in polynucleotide order–disorder transitions is extended to cases where one or more of the participating species is titrated to some degree α. It is shown that, for any class of transition, the melting temperature T_m at constant pH is a linear function of the logarithm of the monovalent counterion concentration M, that at high salt the logarithm of the depression of the melting temperature by pH titration is proportional to the pH change, and that the stability of the ordered form as measured by its melting temperature at neutral pH, is a monotonic function of the quantity pH_m – pK, where pH_m and pK are the pH of melting and the monomer base pK, both measured under similar conditions of temperature and ionic strength. For the transition from double helix to coil, the dependences of T_m and dT_m/d log M on pH are determined experimentally and compared with the qualitative predictions of the theory. It is found that dT_m/dlog M, a measure of – ΔF?^el (the negative of the electrostatic free energy change in the transition), decreases with increasing pH. In acid solution, where the coil is more extensively prolonated than the helix, the change in electrostatic free energy in the transition is larger than at neutral pH. Conversely, in alkali the electrostatic five energy change is smaller than at neutral pH. Hence (dT_m/d log M)_acid > (dT_m/d log M _neutral) > (dT_m/d log M)_alkali. At Suffeciently high pH, dT_m/d log M is observed to become negative, indicating that the electrostatic free energy change is positive in the transition of this region. Date from the literature on the ionic strength dependence of the melting temperature for the acid helices of poly rA, poly rC, and poly dC are also considered from the standpoint of the theory.     

An NMR-based docking model for the physiological transient complex between cytochrome f and cytochrome c6

The physiological transient complex between cytochrome f (Cf) and cytochrome c₆ (Cc₆) from the cyanobacterium Nostoc sp. PCC 7119 has been analysed by NMR spectroscopy. The binding constant at low ionic strength is 8 ± 2 mM⁻¹, and the binding site of Cc₆ for Cf is localized around its exposed haem edge. On the basis of the experimental data, the resulting docking simulations suggest that Cc₆ binds to Cf in a fashion that is analogous to that of plastocyanin but differs between prokaryotes and eukaryotes.     

Comparative proteomics analysis of the effect of combined red and blue lights on sugarcane somatic embryogenesis

Sugarcane is a renewable energy source that provides an alternative to fossil fuels and somatic embryogenesis can improve plant breeding for this purpose. Sugarcane somatic embryo development involves several unknown biochemical and molecular processes, and shotgun proteomics analysis may be helpful for the identification of somatic embryo-related proteins. The aims of this study were to evaluate the influence of combinations of red and blue wavelengths from LED lamps on the maturation of somatic embryos of sugarcane cv. SP80-3280 and to identify proteins that might be associated with the morphogenetic response. Embryogenic callus were subjected to maturation treatments with various combinations of red and blue wavelengths, and a fluorescent lamp was used as a control. The W_mB_dR_fR (450/530/660/735 nm) treatment for 28 days yielded 58 somatic embryos per callus, whereas the control yielded 23 somatic embryos per callus. Samples from the control and W_mB_dR_fR treatments were subjected to shotgun proteomic analysis and the proteomics data were processed with Progenesis QI software against the SUCEST project database. The quantitative shotgun proteomics analysis identified 1171 proteins common to the fluorescent and W_mB_dR_fR treatments, and of these 39 and 38 proteins were up-regulated and down-regulated, respectively, in the W_mB_dR_fR compared with the fluorescent treatment. The W_mB_dR_fR treatment induced a higher abundance of methyltransferases and clathrin heavy chain 1, which are related to differentiation and dedifferentiation processes and might be candidate markers for sugarcane somatic embryogenesis. Overall, our data indicate that LEDs can be advantageous substitutes for fluorescent lamps and that a combination of red and blue lights can enhance somatic embryo maturation and conversion in sugarcane.     

Water permeability of thin lipid membranes

The osmotic permeability coefficient, P_f, and the tagged water permeability coefficient, P_d, were determined for thin (<100 A) lipid membranes formed from ox brain lipids plus DL-α-tocopherol; their value of approximately 1 x 10^-3 cm/sec is within the range reported for plasma membranes. It was established that P_f = P_d. Other reports that P_f > P_d can be attributed to the presence of unstirred layers in the experimental determination of P_d. Thus, there is no evidence for the existence of aqueous pores in these thin phospholipid membranes. The adsorption onto the membrane of a protein that lowers its electrical resistance by a factor of 10³ was found not to affect its water permeability; however, glucose and sucrose were found to interact with the membrane to modify P_f. Possible mechanisms of water transport across these films are discussed, together with the implications of data obtained on these structures for plasma membranes.     

Analysis of cooperativity and ion effects in the interaction of quinacrine with DNA

The interaction of quinacrine with calf thymus DNA was monitored at several different ionic strengths using spectrophotometric and equilibrium dialysis techniques. The binding results can be explained, assuming each base pair is a potential binding site, using a model containing two negative cooperative effects: (1) ligand exclusion at binding sites adjacent to a filled binding site and (2) ligand–ligand negative cooperativity at adjacent filled binding sites. The logarithm of the observed equilibrium constant (K_obs) determined by this model varies linearily with log[Na⁺], as predicted by the ion condensation theory for polyelectrolytes. When the log K_obs plot is correlated for sodium release by DNA in the intercalation conformational change, the predicted number of ion pairs between the ligand and DNA is approximately two, as expected for the quinacrine dication. Even though K_obs depends strongly on ionic strength, the ligand negative cooperativity parameter ω was found to be indpendent of ionic strength within experimental error. This finding is also in agreement with the ion condensation theory, which predicts a relatively constant amount of condensed counterion on the DNA double helix over this ionic strength range. Drugs would, therefore, experience a relatively constant ionic environment when complexed to DNA even though the ionic conditions of the solvent could change considerably.     

Changes in Ultrastructure and Subunit Composition of Protein Body in Rice Endosperm during Germination

Changes in ultrastructure of protein bodies in subaleurone cells of rice endosperm during germination were studied by transmission electron microscopy. The subaleurone cells contained two different types of protein bodies: PB-I (spherical) and PB-II (crystalline). Both types of protein bodies were deconstructed during germination. But there was a considerable difference in digestibility between PB-I and PB-II. PB-II which did not have a dense core was easily digested from the central portion when germination began. At 6 days of germination, PB-II was almost deconstructed. On the other hand, PB-I which displayed concentric rings with a dense core was digested from the outside after 3 days of germination. At 9 days of germination, many kernels of the spherical protein bodies remained.

Changes in subunit composition of protein bodies during germination were investigated by SDS-polyacrylamide gel electrophoresis. Protein body fractions were isolated from germinating grains at various stages by enzymatic digestion and two-phase system, then subjected to SDS-polyacrylamide gel. As germination proceeded, 15 (b₁), 20 (d₁), 24 (e), 35 (f₁) and 37 (f₃) kdaltons subunits decreased. On the other hand, 16 (b₂), 21 (d₂) and 36 (f₂) k daltons subunits remained at the later stage of germination. We think that PB-I contains b₂, d₂ and f₂ subunits and is attacked only from the outside at middle and later stages of germination by de novo protease. On the contrary, PB-II contains b₁ d₁ e, f₁ and f₃ subunits is utilized at an early stage of germination. PB-II may possibly contain latent protease. The breakdown process of PB-I was by exo-type digestion, on the contrary, that of PB-II was by endo-type digestion.     

Multiparticle computer simulation of plastocyanin diffusion and interaction with cytochrome <Emphasis Type="Italic">f</Emphasis> in the electrostatic field of the thylakoid membrane

A multiparticle computer model of plastocyanin-cytochrome f complex formation in the thylakoid lumen has been designed, which takes into account the electrostatic interactions of proteins and membrane. The Poisson-Boltzmann formalism was used to determine the electrostatic potentials of the electron carrier proteins and the thylakoid membrane at different ionic strengths. The membrane electrostatic field was shown to influence plastocyanin diffusion and interaction with cytochrome f. The rate constants for plastocyanin-cytochrome f complex formation were calculated as a function of ionic strength and membrane surface charge.     

Ionic strength effect on adsorption of cellobiohydrolases I and II on microcrystalline cellulose

Cellobiohydrolase I (CBH I) has a higher adsorption affinity (K _ad) and tightness (–H _a) for Avicel than cellobiohydrolase II (CBH II). The adsorption processes of CBH I and II were exothermic, and the degree of exothermy were larger with the increasing ionic strength. Entropy change of CBH I was larger than CBH II with increasing ionic strength. CBH I was more effective than CBH II for binding at a given ionic strength.     

Helix-coil transition in nucleoprotein. Effect of ionic strength on thermal denaturation of polylysine-DNA complexes

Thermal denaturation of direct-mixed and reconstituted polylysine–DNA complexes in 2.5 × 10^?4 M EDTA, pH 8.0 and various concentrations of NaCl has been studied. For both complexes, increasing ionic strength of the solution raises T_m, the melting temperature of free base pairs. The linear dependence of T_m on log Na⁺ indicates that the concept of electrostatic shielding on phosphate lattice of an infinitely long pure DNA by Na⁺ can be applied to short free DNA segments in a nucleoprotein. For a direct-mixed polylysine–DNA complex, the melting temperature of bound base pairs T_m′ remains constant at various ionic strengths. On the other hand, the T_m′ in a reconstituted polylysine–DNA complex is shifted to lower temperature at higher ionic strength. This phenomenon occurs for reconstituted complex with long polylysine of one thousand residues or short polylysine of one hundred residues. It is shown that such a decrease of T_m′ is not due to a reduction of coupling melting between free and bound regions in a complex when the ionic strength is raised. It is also not due to intermolecular or intramolecular change from a reconstituted to a direct-mixed complex. It is suggested that this phenomenon is due to structural change on polylysine-bound regions by ionic strength. It is suggested further that Na⁺ may replace water molecules and bind polylysine-bound regions in a reconstituted complex. Such a dehydration effect destabilizes these regions and lowers T_m′. This explanation is supported by circular dichroism (CD) results.     

Osmotic Pressure of DNA Solutions and Effective Diameter of the Double Helix

Abstract

A simple osmometer with nuclear filters (polymer films with pores of a preset diameter) were used to measure the osmotic pressure of Col El plasmid DNA solutions in the concentration range of 1–4 mg/ml DNA. Linear and open circular DNA forms proved to have the same osmotic pressure within the experimental accuracy. The results of the measurements were used for calculating the second virial coefficient A ₂ of the solution of DNA segments and the effective chain diameter d _eff in the ionic strength range of 10^?2-0.1 M, As the ionic strength is lowered from 0.1 to 10^?2 M the effective diameter of DNA increases from 80 to 220 A. The results are in rather good agreement with theory and with other experimental data.