Pavel I. Semenyuk

Improving the dispersity of detonation nanodiamond: differential scanning calorimetry as a new method of controlling the aggregation state of nanodiamond powders

Detonation nanodiamond (ND) is a suitable source material to produce unique samples consisting of almost uniform diamond nanocrystals (d = 3-5 nm). Such samples exist in the form of long stable aqueous dispersions with narrow size distribution of diamond particles. The material is finding ever increasing application in biomedicine. The major problem in producing monodispersed diamond colloids lies in the necessity of deagglomeration of detonation soot and/or removing of clusters formed by already isolated core particles in dry powders. To do this one must have an effective method to monitor the aggregation state or dispersity of powders and gels prior to the preparation of aqueous dispersions. In the absence of dispersity control at various stages of preparation the reproducibility of properties of existing ND materials is poor. In this paper we introduce differential scanning calorimetry (DSC) as a new tool capable to distinguish the state of aggregation in dry and wetted ND materials and to follow changes in this state under different types of treatment. Samples with identical X-ray diffraction patterns (XRD) and high resolution transmission electron microscopy (HRTEM) images gave visibly different DSC traces. Strong correlation was found between dynamic light scattering (DLS) data for colloids and DSC parameters for gels and powders of the same material. Based on DSC data we improved dispersity of existing ND materials and isolated samples with the best possible DSC parameters. These were true monodispersed easily dispersible fractions of ND particles with diameters of ca. 3 nm.

Expression and Functional Characterization of the First Bacteriophage-Encoded Chaperonin

ABSTRACT Chaperonins promote protein folding in vivo and are ubiquitously found in bacteria, archaea, and eukaryotes. The first viral chaperonin GroEL ortholog, gene product 146 (gp146), whose gene was earlier identified in the genome of bacteriophage EL, has been shown to be synthesized during phage propagation in Pseudomonas aeruginosa cells. The recombinant gp146 has been expressed in Escherichia coli and characterized by different physicochemical methods for the first time. Using serum against the recombinant protein, gp146s native substrate, the phage endolysin gp188, has been immunoprecipitated from the lysate of EL-infected bacteria and identified by mass spectrometry. In vitro experiments have shown that gp146 has a protective effect against endolysin thermal inactivation and aggregation, providing evidence of its chaperonin function. The phage chaperonin has been found to have the architecture and some properties similar to those of GroEL but not to require cochaperonin for its functional activity.

GAPDH binders as potential drugs for the therapy of polyglutamine diseases: design of a new screening assay.

Proteins with long polyglutamine repeats form a complex with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), which enhances aggregation and cytotoxicity in models of Huntington disease. The aim of this study was to develop a novel assay for the screening of anti‐aggregation compounds with a focus on the aggregation‐promoting capacity of GAPDH. The assay includes a pure Q58 polyglutamine fragment, GAPDH, and a transglutaminase that links the two proteins. The feasibility of the new assay was verified using two GAPDH binders, hydroxynonenal and −(−)deprenyl, and the benzothiazole derivative PGL‐135 which exhibits anti‐aggregation effect. All three substances were shown to reduce aggregation and cytotoxicity in the cell and in the fly model of Spinocerebellar ataxia.

Effect of 2-hydroxypropyl-β-cyclodextrin on thermal inactivation, denaturation and aggregation of glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscle.

The effect of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on thermal aggregation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle at 45 degrees C has been studied using dynamic light scattering. In the presence of HP-beta-CD higher values of the rate of aggregation and larger aggregates were registered. The acceleration of GAPDH aggregation was due to destabilization of the enzyme molecule under the action of HP-beta-CD. This is evidenced by the data on thermal inactivation of GAPDH and differential scanning calorimetry.

Sulfated and sulfonated polymers are able to solubilize efficiently the protein aggregates of different nature

The search for new ways to suppress unwanted protein aggregation represents an important problem in modern biochemistry, bioengineering, and even medicine. Recently we succeeded in preventing the aggregation using synthetic polyelectrolytes. The present work describes a new approach to solubilizing pre-formed protein aggregates with sulfated or sulfonated polymers (polysulfoanions). For the first time it was shown that polysulfoanions are capable of solubilizing amorphous and amyloid protein inclusion bodies as well as thermal aggregates. Treatment of prion protein inclusion bodies with sulfonated polymers was shown to cause significant decrease in amyloid structure content, whereas in case of thermal aggregates of glyceraldehyde-3-phosphate dehydrogenase the observed solubilization was accompanied by a partial recovery of enzymatic activity. The suggested approach could be relevant in the task of extracting recombinant proteins from inclusion bodies and also useful in the development of amyloid disease therapy.

Effect of poly(phosphate) anions on glyceraldehyde-3-phosphate dehydrogenase structure and thermal aggregation: comparison with influence of poly(sulfoanions)

BACKGROUND It is well documented that poly(sulfate) and poly(sulfonate) anions suppress protein thermal aggregation much more efficiently than poly(carboxylic) anions, but as a rule, they denature protein molecules. In this work, a polymer of different nature, i.e. poly(phosphate) anion (PP) was used to elucidate the influence of phosphate groups on stability and thermal aggregation of the model enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). METHODS Isothermal titration calorimetry and differential scanning calorimetry were used for studying the protein-polyanion interactions and the influence of bound polyanions on the protein structure. The enzymatic activity of GAPDH and size of the complexes were measured. The aggregation level was determined from the turbidity. RESULTS Highly polymerized PP chains were able to suppress the aggregation completely, but at significantly higher concentrations as compared with poly(styrenesulfonate) (PSS) or dextran sulfate chains of the same degree of polymerization. The effect of PP on the enzyme structure and activity was much gentler as opposed to the binding of dextran sulfate or, especially, PSS that denatured GAPDH molecules with the highest efficacy caused by short PSS chains. These findings agreed well with the enhanced affinity of polysulfoanions to GAPDH. CONCLUSIONS The revealed trends might help to illuminate the mechanism of control of proteins functionalities by insertion of charged groups of different nature through posttranslational modifications. GENERAL SIGNIFICANCE Practical implementation of the results could be the use of PP chains as promising tools to suppress the proteins aggregation without noticeable loss in the enzymatic activity.

Structural lability of Barley stripe mosaic virus virions.

Virions of Barley stripe mosaic virus (BSMV) were neglected for more than thirty years after their basic properties were determined. In this paper, the physicochemical characteristics of BSMV virions and virion-derived viral capsid protein (CP) were analyzed, namely, the absorption and intrinsic fluorescence spectra, circular dichroism spectra, differential scanning calorimetry curves, and size distributions by dynamic laser light scattering. The structural properties of BSMV virions proved to be intermediate between those of Tobacco mosaic virus (TMV), a well-characterized virus with rigid rod-shaped virions, and flexuous filamentous plant viruses. The BSMV virions were found to be considerably more labile than expected from their rod-like morphology and a distant sequence relation of the BSMV and TMV CPs. The circular dichroism spectra of BSMV CP subunits incorporated into the virions, but not subunits of free CP, demonstrated a significant proportion of beta-structure elements, which were proposed to be localized mostly in the protein regions exposed on the virion outer surface. These beta-structure elements likely formed during virion assembly can comprise the N- and C-terminal protein regions unstructured in the non-virion CP and can mediate inter-subunit interactions. Based on computer-assisted structure modeling, a model for BSMV CP subunit structural fold compliant with the available experimental data was proposed.

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase is stabilized by additional proline residues and an interdomain salt bridge.

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) exhibits enhanced stability compared to the somatic isoenzyme (GAPD). A comparative analysis of the structures of these isoenzymes revealed characteristic features, which could be important for the stability of GAPDS: six specific proline residues and three buried salt bridges. To evaluate the impact of these structural elements into the stability of this isoenzyme, we obtained two series of mutant GAPDS: 1) six mutants each containing a substitution of one of the specific prolines by alanine, and 2) three mutants each containing a mutation breaking one of the salt bridges. Stability of the mutants was evaluated by differential scanning calorimetry and by their resistance towards guanidine hydrochloride (GdnHCl). The most effect on thermostability was observed for the mutants P326A and P164A: the Tm values of the heat-absorption curves decreased by 6.0 and 3.3°C compared to the wild type protein, respectively. The resistance towards GdnHCl was affected most by the mutation D311N breaking the salt bridge between the catalytic and NAD(+)-binding domains: the inactivation rate constant in the presence of GdnHCl increased six-fold, and the value of GdnHCl concentration corresponding to the protein half-denaturation decreased from 1.83 to 1.35M. Besides, the mutation D311N enhanced the enzymatic activity of the protein two-fold. The results suggest that the residues P164 (β-turn), P326 (first position of α-helix), and the interdomain salt bridge D311-H124 are significant for the enhanced stability of GAPDS. The salt bridge D311-H124 enhances stability of the active site of GAPDS at the expense of the catalytic activity.

Quantification of nanodiamonds in aqueous solutions by spectrophotometry and thermal lens spectrometry

Spectrophotometry and thermal lens spectrometry were used to study solutions of several commercial detonation nanodiamonds. It was found that the absorption spectra of solutions of all studied nanodiamond samples obey the Bouger-Lambert-Beer law, which ensures the precise determination of the total mass concentration of unknown nanodiamond solutions using a calibration plot. It was shown that the absorption spectra of nanodiamond solutions exhibit both absorption and scattering components, both significantly affecting signal formation. Conditions were proposed for the spectrophotometric determination of nanodiamonds at 250 nm (l = 1.0 cm). The detection limits were from 60 ng/mL to 2 μg/mL, depending on the nanodiamond type. Limits of detection of SDND nanodiamonds by spectrophotometry and thermal lens spectrometry were calculated for identical conditions of sample preparation and measurements (488 nm, l = 1.0 cm). These were 10 and 0.6 μg/mL for spectrophotometry and thermal lens spectrometry, respectively (power of excitation radiation 150 mW).

Partially disordered structure in intravirus coat protein of potyvirus potato virus A.

Potyviruses represent the most biologically successful group of plant viruses, but to our knowledge, this work is the first detailed study of physicochemical characteristics of potyvirus virions. We measured the UV absorption, far and near UV circular dichroism spectra, intrinsic fluorescence spectra, and differential scanning calorimetry (DSC) melting curves of intact particles of a potato virus A (PVA). PVA virions proved to have a peculiar combination of physicochemical properties. The intravirus coat protein (CP) subunits were shown to contain an unusually high fraction of disordered structures, whereas PVA virions had an almost normal thermal stability. Upon heating from 20°C to 55°C, the fraction of disordered structures in the intravirus CP further increased, while PVA virions remained intact at up to 55°C, after which their disruption (and DSC melting) started. We suggest that the structure of PVA virions below 55°C is stabilized by interactions between the remaining structured segments of intravirus CP. It is not improbable that the biological efficiency of PVA relies on the disordered structure of intravirus CP.