Time-resolved fluorescence analysis of the mobile flavin cofactor in p -hydroxybenzoate hydroxylase

Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the ‘in’ conformation with the isoalloxazine ring located in the active site, and the ‘out’ conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the ‘in’ and ‘out’ conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes.     

Synthesis, characterization and protein adsorption behaviors of PLGA/PEG di-block co-polymer blend films

A series of poly(D,L-lactic-co-glycolic acid) (PLGA)/poly(ethyleneglycol) (PEG) di-block copolymers were synthesized by ring-opening polymerization of D,L-lactide and glycolide with different molecular weights of monomethoxy polyethyleneglycol (mPEG) 750, 2000 and 5000 as an initiator. The bulk properties of these co-polymers were characterized by using 1H NMR spectroscopy, gel permeation chromatography, differential scanning calorimetry (DSC). Electron spectroscopy for chemical analysis (ESCA) results, in which the blend films with the di-block copolymers showed increasing surface oxygen atomic percentage with increasing PEG chain length, indicate that PEG chain segment in the di-block copolymers is surface oriented and enriched onto the surface of the blend films. The extent of protein adsorption onto the surface of these blend films was studied, using iodine radio-labeled human serum albumin, gamma globulin and human growth hormone. The protein adsorption amount was reduced for the blend films prepared with PLGA/PEG 750 and 2000 di-block copolymers, but increased to a great extent for PLGA/PEG 5000 di-block copolymer. This is due to the increased water uptake capacity of the blend film, which absorbed more protein molecules into a swollen polymer matrix in addition to surface adsorption.     

A new method for reversible immobilization of thiol biomolecules based on solid-phase bound thiolsulfonate groups

A new method for the reversible immobilization of thiol bimolecules, e.g., thiolpeptides and thiolproteins, to beaded agarose and other solid phases is reported. The method consists of an activation and a coupling step. The activation is based on oxidation of disulfides (or thiol groups via disulfides) present in a solid phase by hydrogen peroxide at moderately acidic pH. This oxidation leads to disulfide oxides (thiolsulfinate groups of which the majority are further oxidized to thiolsulfonate). The thiolsulfonate groups react easily with thiol compounds, which become immobilized via disulfide bonds. The pH range for thiol coupling is wide (pH 5-8), but for most thiols the reaction seems to proceed faster at pH>7. The stability of the reactive group to hydrolysis, especially at neutral and weakly acidic pH, is very high. The activated gel, therefore, can be stored as a suspension at pH 5 for extended periods. The method has been used to reversibly immobilize glutathione, β-galactosidase, alcohol dehydrogenase, urease, and papain, all with exposed thiol groups as well as thiolated bovine serum albumin and sweet-potato β-amylase. Depending on the thiol content of starting thiol-agarose, thiol-sulfonate-agarose derivatives with different binding capacities can be obtained. Thus, up to 5.0 mg (16 μmol) glutathione and 15 mg thiol-protein/mL gel derivative have been immobilized.     

Influence of the aggregation state in solution on the supramolecular organization of adsorbed type I collagen layers

In the last years, adsorbed collagen was shown to form layers with a supramolecular organization depending on the substrate surface properties and on the preparation procedure. If the concentration of collagen and the duration of adsorption are sufficient, fibrillar collagen structures are formed, corresponding to assemblies of a few molecules. This occurs more readily on hydrophobic compared to hydrophilic surfaces. This study aims at understanding the origin of such fibrillar structures and in particular at determining whether they result from the deposition of fibrils formed in solution or from the building of assemblies at the interface. Therefore, type I collagen solutions with an increasing degree of aggregation were prepared, using the “neutral-start” approach, by ageing pH 5.8 solutions at 37 °C for 15 min, 2 or 7 days. The obtained solutions were used to investigate the influence of collagen aggregation in solution on the supramolecular organization of adsorbed collagen layers, which was characterized by X-ray photoelectron spectroscopy and atomic force microscopy. Polystyrene and plasma-oxidized polystyrene were chosen as substrates for the adsorption. The size and the density of collagen fibrils at the interface decreased upon increasing the degree of aggregation of collagen in solution. This is explained by a competitive adsorption process between monomers and aggregates of the solution, turning at the advantage of the monomers. More aggregated solutions, which are thus depleted in free monomers, behave like less concentrated solutions, i.e. lead to a lower adsorbed amount and less fibril formation at the interface. This study shows that the supramolecular fibrils observed in adsorbed collagen layers, especially on hydrophobic substrates, are not formed in the solution, prior to adsorption, but are built at the interface, through the assembly of free segments of adsorbed molecules.     

Influence of high pressure treatment on rheological and other properties of egg white

Abstract

The paper deals with the influence of high pressure treatment of fresh egg white on its properties and protein composition (individual amino-acids predicted as a function of pressure and time levels). The rheological properties are changed by high pressure from Newtonian to non-Newtonian behaviour, with increasing apparent viscosity as the pressure and time increased. The pH, whipping ability, foam stability, gel strength of heat induced gels after treatment and the whole protein content, were also predicted.

The results showed that the foam stability is increased with increasing pressure and time of processing. The foam volume is also increased with pressure. The pH did not change with pressure or time of processing. Composition of proteins as indicated by individual amino-acids did not exhibit statistically important changes. Gel strength of heat induced gels prepared from previously pressured liquid whites showed no important change of values with pressure or time of treatment. The modulus of elasticity showed a decrease for samples pressured to 400 MPa for 5 up to 15 minutes.     

Effect of purified fractions from cell culture supernate of high‐density pre‐B acute lymphoblastic leukemia cells (ALL3) on the growth of ALL3 cells at low density

The mechanisms underlying the aberrant growth and interactions between cells are not understood very well. The pre‐B acute lymphoblastic leukemia cells directly obtained from an adult patient grow very poorly or do not grow at all at low density (LD), but grow better at high starting cell density (HD). We found that the LD ALL3 cells can be stimulated to grow in the presence of diffusible, soluble factors secreted by ALL3 cells themselves growing at high starting cell density. We then developed a biochemical purification procedure that allowed us to purify the factor(s) with stimulatory activity and analyzed them by nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS). Using nanoLC‐MS/MS we have identified several proteins which were further processed using various bioinformatics tools. This resulted in eight protein candidates which might be responsible for the growth activity on non‐growing LD ALL3 cells and their involvement in the stimulatory activity are discussed.     

Conformational landscapes of bimesogenic compounds and their implications for the formation of modulated nematic phases

ABSTRACT

The twist-bend phase (N_TB) is most commonly observed in materials with a gross-bent shape: dimers; bent-cores; bent-oligomers. We had suggested previously that the bend-angle of such systems effectively dictates the relative thermal stability of the N_TB phase. However, our earlier paper relied on the use of a single energy-minimum conformer and so failed to capture any information about flexibility and conformational distribution. In the present work, we revisit our hypothesis and examine a second set of dimers with varying linking groups and spacer composition. We have improved on our earlier work by studying the conformational landscape of each material, allowing average bend-angles to be determined as well as the conformer distribution. We observe that the stability of the N_TB phase exhibits a strong dependence not only on the Boltzmann-weighted average bend-angle (rather than just a static conformer), but also on the distribution of conformers. To a lesser extent, the flexibility of the spacer appears important. Ultimately, this work satisfies both theoretical treatments and our initial experimental study and demonstrates the importance of molecular bend to the N_TB phase.     

Modelling toxin effects on protein biosynthesis in eukaryotic cells

We present a rather generic model for toxin (ricin) inhibition of protein biosynthesis in eukaryotic cells. We also study reduction of the ricin toxic effects with application of antibodies against the RTB subunit of ricin molecules. Both species initially are delivered extracellularly. The model accounts for the pinocytotic and receptor-mediated toxin endocytosis and the intact toxin exocytotic removal out of the cell. The model also includes the lysosomal toxin destruction, the intact toxin motion to the endoplasmic reticulum (ER) for separation of its molecules into the RTA and RTB subunits, and the RTA chain translocation into the cytosol. In the cytosol, one portion of the RTA undergoes degradation via the ERAD. The other its portion can inactivate ribosomes at a large rate. The model is based on a system of deterministic ODEs. The influence of the kinetic parameters on the protein concentration and antibody protection factor is studied in detail.