Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing

A continuous-flow mixing device with a dead time of 100 micros coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal beta-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT(*) SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step ( approximately 100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 micros. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the beta-barrel domain while the specific interactions in the alpha-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal beta-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT(*) SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.     

Y97V substitution in the horse cytochrome c causes accumulation of the equilibrium intermediate

Equilibrium unfolding experiments on several mutant forms of horse heart cytochrome c were performed. By means of absorbance spectroscopy, the accumulation of an equilibrium intermediate was revealed upon unfolding of Y97V mutant protein, and its structural properties were characterized. The data obtained allow one to conclude that the equilibrium intermediate corresponds to the earliest kinetic intermediate Ic in cytochrome c folding reaction. A comparative analysis of spectral properties of unfolded states of cytochrome c induced by urea or guanidine hydrochloride is presented.     

Changes in the bioelectrical activity of the brain cortex in rats with infraorbital nerve compression]

Bioelectrical activity of the somatosensory cortex was studied in the Wistar rats with chronic (1.5-2 months) compression of the infraorbital nerve produced by two partial ligations. In 20% of rats spike-slow wave complex and slow waves were observed. Electrostimulation of the skin on the injured nerve side resulted in a considerable increase in the amplitude of early components of the contralaterally evoked potentials in comparison with the non-injured side stimulation in 75% of rats. A decrease in the evoked potential thresholds on the injured nerve stimulation was shown in both hemispheres. In most of the animals a hypersynchronous late component of the evoked potential was observed.     

Effects of carnosine on systemic hemodynamics and myocardial metabolism in rats in the early postresuscitation period]

It was demonstrated in experiments on male rats that acute lethal blood loss and subsequent resuscitation after 4- and 6-min clinical death induce lipid peroxidation processes, decreased antioxidant enzyme activity, cause activation of anaerobic glycolysis in the myocardium. This metabolic heart impairment causes hemodynamic instability in postresuscitation period. 25 mg/kg of carnosine injected during resuscitation decreased functional-metabolic heart impairments and hemodynamic disarrangement as well as early postresuscitation lethality. The authors attribute positive carnosine effect to its significant antioxidant activity.     

Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: Exploring a new role of haptoglobin in the tumoral microenvironment

Haptoglobin (Hp) is an acute-phase protein that is produced by the liver to capture the iron that is present in the blood circulation, thus avoiding its accumulation in the blood. Moreover, Hp has been detected in a wide variety of tissues, in which it performs various functions. In addition, this protein is considered a potential biomarker in many diseases, such as cancer, including ovarian carcinoma; however, its participation in the cancerous processes has not yet been determined. The objective of this work was to demonstrate the expression of Hp and its receptor CCR2 in the ovarian cancer cells and its possible involvement in the process of cell migration through changes in the rearrangement of the actin cytoskeleton using western blot and wound-healing assays and confirming by confocal microscopy. Ovarian cancer cells express both Hp and its receptor CCR2 but only after exposure to ascitic fluid, inducing moderated cell migration. However, when the cells are exposed to exogenous Hp, the expression of CCR2 is induced together with drastic changes in the actin cytoskeleton rearrangement. At the same time, Hp induced cell migration in a much more efficient manner than did ascitic fluid. These effects were blocked when the CCR2 synthetic antagonist RS102895 was used to pretreat the cells. These results suggest that Hp-induced changes in the cell morphology, actin cytoskeleton structure, and migration ability of tumor cells, is possibly “preparing” these cells for the potential induction of the metastatic phenotype.     

Patterns of ISSR and REMAP DNA markers after cryogenic preservation of spring wheat calli by dehydration method

Inter-Simple Sequence Repeat (ISSR) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers were applied to study the influence of successive steps of dehydration cryopreservation on DNA in recovered calli and regenerated plants of spring wheat (Triticum aestivum L.). The precultivation step had no influence on the genetic stability of plant material. After the dehydration step, a new fragment appeared in the REMAP profiles for one DNA sample of calli of Nv16 line. A fragment of similar length was observed in one DNA sample for calli regenerated after complete procedure of cryopreservation in liquid nitrogen (−196°C). However, in samples of calli cultured in vitro for two and four weeks after any type of treatments, the amplicon spectra exhibited no difference from those of starting materials. The amplicon profiles of plants regenerated from calli after successive steps of cryopreservation were also identical to the profiles of the mother plants.     

Study of Interaction of Fluorescent Cytochrome C with Liposomes,Mitochondria, and Mitoplasts by Fluorescence Correlation Spectroscopy

Russian Journal of Bioorganic Chemistry - Here, we studied the interaction of Cys-substituted (G56C) cytochrome c labeled with sulfocyanin-3 fluorescent dye (fCyt) with artificial and natural lipid...     

Development of a Pilot Technology for the Production of the Recombinant Human Enteropeptidase Light Chain in Soluble and Immobilized Forms

Russian Journal of Bioorganic Chemistry - The proteolytic enzyme enteropeptidase (EC 3.4.21.9) is a glycoprotein consisting of a heavy and a light polypeptide chain. The light chain of the enzyme...     

Alternative scaffold proteins

Review is devoted to the challenging direction in modem molecular biology and bioengineering - the properties of alternative scaffold proteins (ASP) and methods for obtaining ASP binding molecules. ASP molecules incorporate conservative protein core and hypervariable regions, providing for the binding function. Structural classification of ASP includes several types which differ also in their molecular targets and potential applications. Construction of artificial binding proteins on the ASP basis implies a combinatorial library design with subsequent selection of specific binders with the use of phage display or the modem cell-free systems. Alternative binding proteins on non-immunoglobulin scaffolds find broad applications in different fields ofbiotechnology and molecular medicine.