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Traditional antiviral vaccines are currently created by inactivating the virus chemically, most often using formaldehyde or β-propiolactone. These approaches are not optimal since they negatively affect the safety of the antigenic determinants of the inactivated particles and require additional purification stages. The most promising platforms for creating vaccines are based on pseudoviruses, i.e., viruses that have completely preserved the outer shell (capsid), while losing the ability to reproduce owing to the destruction of the genome. The irradiation of viruses with electron beam is the optimal way to create pseudoviral particles. In this review, with the example of the poliovirus, the main algorithms that can be applied to characterize pseudoviral particles functionally and structurally in the process of creating a vaccine preparation are presented. These algorithms are, namely, the analysis of the degree of genome destruction and coimmunogenicity. The structure of the poliovirus and methods of its inactivation are considered. Methods for assessing residual infectivity and immunogenicity are proposed for the functional characterization of pseudoviruses. Genome integrity analysis approaches, atomic force and electron microscopy, surface plasmon resonance, and bioelectrochemical methods are crucial to structural characterization of the pseudovirus particles.

We have isolated fractions of 26S and 20S proteasomes were from the rabbit liver and the brain. According to mass spectrometric (MS) analysis, the 26S proteasome fractions from these organs contained catalytic and regulatory subunits characteristic of the proteasome core and regulatory subunits. The 20S fractions of brain and liver proteasomes contained only catalytic proteasome subunits. In addition to proteasome subunits, the isolated fractions contained components of the ubiquitin-proteasome system, ubiquitinated proteins, enzymes that play an important role in metabolic processes, cytoskeletal components, signaling, regulatory, and protective proteins, as well as proteins regulating gene expression, cell division, and differentiation. The abundance of a number of proteasome-associated proteins was comparable or exceeded the abundance of intrinsic proteasome components. About a third of the proteins common to all studied fractions (26S and 20S of brain and liver proteasomes) belong to the group of multifunctional proteins. Selective biosensor validation confirmed the affinity binding of proteins (aldolase, phosphoglycerate kinase) identified during MS analysis to the brain 20S proteasome. Comparison of the subproteomes of the 26S and 20S brain proteasomes showed that removal of components of the regulatory (19S) subparticles caused almost two-fold increase in the total number of individual proteins associated with the core part of the proteasome (20S). In the liver, the number of proteins associated with the core part of the proteasome remained basically unchanged after the removal of the components of the regulatory (19S) subparticles. This indicates that in the brain and, possibly, in other organs, proteins of the regulatory (19S) subunit play an important role in the formation of the proteasome interactome.

The role of partner proteins in the formation of functional complexes in cytochrome P450 systems was investigated by means of optical biosensor technique. Kinetic constants and equilibrium dissociation constants of complexes of cytochrome CYP11A1 (P450scc) with wild-type adrenodoxin (Adx WT) and mutant forms of adrenodoxin R106D and D109R were determined using an optical biosensor. Wild-type adrenodoxin (Kd = (1.23±0.09)⋅10⁻⁶ M) and mutant D109R (Kd = (2.37±0.09)⋅10⁻⁸ M) formed complexes with cytochrome P450scc. For the R106D mutant, no complex formation was detected. To investigate the possibility of the participation of adrenodoxins and their mutant variants in the process of electron transfer as electron donors in mitochondrial cytochrome P450 systems, the electrochemical properties of these iron-sulfur proteins Adx WT and mutant forms of adrenodoxins were studied. Adx WT, mutant forms R106D and D109R have redox potentials E1/2 significantly more negative than cytochromes P450 (-579±10 mV, -590±15 mV, and -528±10 mV, respectively). These results suggest that Adx WT and mutant forms may be electron donors in the cytochrome P450 systems.

Currently, opportunistic fungi of the genus Candida are the main causative agents of mycoses, which are especially severe upon condition of acquired immunodeficiency. The main target for the development of new antimycotics is the cytochrome P450 51 (CYP51) of the pathogenic fungus. Due to the widespread distribution of Candida strains resistancy to inhibitors of the azole class, the screening for CYP51 inhibitors both among non-azole compounds and among clinically used drugs repurposing as antimycotics is becoming urgent. To identify potential inhibitors from the non-azole group, an integrated approach was applied, including bioinformatics analysis, computer molecular modeling, and a surface plasmon resonance (SPR) technology. Using in silico modeling, the binding sites for acetylsalicylic acid, ibuprofen, chlorpromazine and haloperidol (this compounds, according to the literature, showed antimycotic activity) were predicted in the active site of CYP51 of Candida albicans and Candida glabrata. The Kd values of molecular complexes of acetylsalicylic acid, ibuprofen and haloperidol with CYP51, determined by SPR analysis, ranged from 18 μM to 126 μM. It was also shown that structural derivatives of haloperidol, containing various substituents, could be positioned in the active site of CYP51 of Candida albicans with the possible formation of coordination bonds between the hydroxyl groups of the derivatives and the iron atom in the heme of CYP51. Thus, the potential basic structures of non-azole compounds have been proposed, which can be used for the design of new CYP51 inhibitors of Candida fungi.

Mitochondrial dysfunction and ubiquitin-proteasome system (UPS) failure contribute significantly to the development of Parkinson's disease (PD). The proteasome subunit Rpn13 located on the regulatory (19S) subparticle play an important role in the delivery of proteins, subjected to degradation, to the proteolytic (20S) part of proteasome. We have previously found several brain mitochondrial proteins specifically bound to Rpn13 (Buneeva et al. (2020) Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 14, 297-305). In this study we have investigated the effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroprotector isatin on the mitochondrial subproteome of Rpn13-binding proteins of the mouse brain. Administration of MPTP (30 mg/kg) to animals caused movement disorders typical of PD, while pretreatment with isatin (100 mg/kg, 30 min before MPTP) reduced their severity. At the same time, the injection of MPTP, isatin, or their combination (isatin + MPTP) had a significant impact on the total number and the composition of Rpn13-binding proteins. The injection of MPTP decreased the total number of Rpn13-binding proteins in comparison with control, and the injection of isatin prior to MPTP or without MPTP caused an essential increase in the number of Rpn13-binding proteins, mainly of the functional group of proteins participating in the protein metabolism regulation, gene expression, and differentiation. Selected biosensor validation confirmed the interaction of Rpn13 subunit of proteasome with some proteins (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, histones H2A and H2B) revealed while proteomic profiling. The results obtained testify that under the conditions of experimental MPTP-induced parkinsonism the neuroprotective effect of isatin may be aimed at the interaction of mitochondria with the components of UPS.

The aim of the present work was to establish the thermodynamic and functional differences in the protein-protein interactions between the components of the P450-dependent mitochondrial (mit) and microsomal (mic) monooxygenase systems using 12 different isoforms of cytochromes P450 and two redox partners, NADPH-dependent cytochrome P450 reductase (CPR) and adrenodoxin (Adx). Comparative analysis of the affinity, thermodynamics, enzymatic activity and the ability for one-electron reduction has been carried out. The study of protein-protein interactions to determine the equilibrium dissociation constants (Kd) was performed using surface plasmon resonance (SPR) biosensor Biacore 3000. We demonstrated that CPR and Adx interacted with both, micCYPs and mitCYPs, with different affinities (Kd values ranged from 0.01 to 2 μM). All complexes of microsomal (micCYP) and mitochondrial (mitCYP) cytochrome P450 with redox partners can be divided into three groups depending on the prevalent role of either enthalpy or entropy contribution. About 90% of CYP/redox partner complexes were entropy-driven, while the contribution of enthalpy and entropy differed significantly in case of mitCYP/Adx complexes. The CYP11A1/Adx complex was enthalpy-driven, while CYP11B1/Adx and CYP11B2/Adx complexes were entropy-driven. Thermodynamic discrimination of mitCYPs/Adx complexes is likely associated with the different functional impact of CYP11A1 and CYP11B. The exception was the enthalpy-entropy-driven (mixed type) CYP21A2/Adx complex. CPR and Adx were able to transfer the first electron to micCYPs while mitCYPs demonstrated high specificity to Adx. Productive catalysis for mitCYPs observed only in the presence of Adx/AdR pair, while in case of steroidogenic micCYPs (CYP17A1, CYP19A1, and CYP21A2) it was found either in the presence of a CPR or an Adx/AdR pair. From the evolutionary point of view, the type 1 electron transport system (mitCYPs, Adx and NADPH-dependent adrenodoxin reductase (AdR)) increased the specialization of protein-protein interactions (PPI) significantly, which was accompanied by an increase in the specificity of electron transfer. In contrast, the evolution of the type 2 electron transport system (micCYPs and CPR) led to an increase in versatility of PPI as demonstrated for steroidogenic microsomal cytochrome P450s. Our data enhance the current understanding of molecular recognition and summarize qualitative and thermodynamic characteristics of protein-protein interactions in the P450-dependent mitochondrial and microsomal monooxygenase systems., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

It becomes increasingly clear that ubiquitination of cellular proteins is not an indispensable prerequisite of their degradation in proteasomes. There are a number of proteins to be eliminated which are not pre-ubiquitinated for their recognition by regulatory subcomplex of 26S proteasome, but which directly interact with the 20S proteasome core particle (20S proteasome). The obligatory precondition for such interaction consists in existence of disordered (hydrophobic) fragments in the target protein. In this study we have investigated the interaction of a number of multifunctional (moonlighting) proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase) and neurodegeneration-related proteins (a-synuclein, myelin basic protein) with 20S proteasome immobilized on the SPR-biosensor chip and stabilized by means of a bifunctional agent dimethyl pimelimidate (in order to prevent possible dissociation of this subcomplex). Only two of all investigated proteins (aldolase and pyruvate kinase) interacted with the immobilized 20S proteasome (Kd of 8.17´10-7 M and 5.56´10-7 M, respectively). In addition to earlier detected GAPDH ubiquitination, mass spectrometric analysis of the studied proteins revealed the presence of the ubiquitin signature (Lys-e-Gly-Gly) only in aldolase. Oxidation of aldolase and pyruvate kinase, which promotes elimination of proteins via their direct interaction with 20S proteasome, caused a 2-3-fold decrease in their Kd values as comparison with this parameter obtained for the intact proteins. The results of this study provide further evidence for direct interaction of both ubiquitinated proteins (aldolase), and non-ubiquitinated proteins (pyruvate kinase) with the 20S proteasome core particle (20S proteasome). The effectiveness of this interaction is basically equal for the ubiquitinated proteins and non-ubiquitinated proteins.

Biosensor experiments on investigation of interaction between prostacyclin synthase (PGIS) and different proteins of the cytochrome P450 monooxygenase systems were perfomed. Interaction of PGIS with microsomal (CYP21A2, CYP2E1) and mitochondrial (CYP27A1, CYP11B1, CYP11B2, CYP11A1) cytochrome P450s was detected. Kinetic and equilibrium parameters of protein complexes formation were determined. Data obtained suggest an essential role of these hemoproteins interaction in regulation of prostacyclin and thromboxane A2 biosynthesis.

Amyloid-β peptide (1-42) (Aβ1-42) is a key player in the development and progression of Alzheimer's disease (AD) and related pathologies, determined by formation of protein aggregates in the central nervous system. Aβ1-42 binding to crucial intracellular targets (and their subsequent inactivation) obviously represents one of the earliest events preceding extracellular pathogenic oligomerization/aggregation of Aβ1-42. It is reasonable to expect that dissociation of the Aβ1-42 complexes with intracellular proteins by means of inhibitors followed by subsequent degradation of Aβ1-42 would not only protect critically important proteins but also prevent intracellular accumulation of Aβ1-42. The aim of this study was to investigate the effect of the neuroprotector isatin (100 mM) on interaction of known Aβ-binding proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase, with Aβ1-42 and its fragments (Aβ1-28, Aβ12-28, Aβ25-35). Aβ1-42 and its fragments (Aβ1-28, Aβ12-28, Aβ25-35) immobilized on the Biacore optical biosensor chip interacted with GAPDH and pyruvate kinase. The lowest and basically equal Kd values were determined for GAPDH and pyruvate kinase complexes with immobilized Aβ1-42 and Aβ25-35. The presence of 100 mM isatin caused a significant (more than fivefold) increase in the Kd values for GAPDH complexes with all Aβ peptides except Aβ1-28. In contrast to GAPDH isatin increased dissociation of pyruvate kinase complexes only with Aβ1-42 (causing a 30-fold increase in Kd) and to a lesser extent with Aβ12-28 and Aβ25-35 (a 10-fold increase in Kd). It should be noted that in the presence of isatin the Kd values for GAPDH and pyruvate kinase complexes with all Aβ studied were in a narrower concentration range (10-7 M - 10-6 M) than in the absence of this neuroprotector (10-8 M - 10-6 M). Data obtained suggest existence of principal possibility of (pharmacological) protection of crucial intracellular targets against both Aβ1-42, and its aggressive truncated peptides (Aβ25-35).

Recent proteomic profiling of mouse brain preparations using the ubiquitin receptor, Rpn10 proteasome subunit, as an affinity ligand revealed a representative group of proteins bound to this sorbent (Medvedev, A. E., et al. (2017) Biochemistry (Moscow), 82, 330-339). In the present study, we investigated interaction of the Rpn10 subunit of proteasomes with some of these identified proteins: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase, and histones H2A and H2B. The study revealed: (i) quantitative affinity interaction of the proteasome subunit immobilized on a Biacore-3000 optical biosensor cuvette with both the GAPDH (K d  = 2.4·10 -6  M) and pyruvate kinase (K d  = 2.8·10 -5  M); (ii) quantitative high-affinity interaction of immobilized histones H2A and H2B with the Rpn10 subunit (K d values of 6.5·10 -8  and 3.2·10 -9  M, respectively). Mass spectrometric analysis revealed the presence of the ubiquitin signature (GG) only in a highly purified preparation of GAPDH. We suggest that binding (especially high-affinity binding) of non-ubiquitinated proteins to the Rpn10 proteasome subunit can both regulate the functioning of this proteasomal ubiquitin receptor (by competing with ubiquitinated substrates) and promote activation of other pathways for proteolytic degradation of proteins destined to the proteasome.

Cytochromes P450 (CYP) are involved in numerous biochemical processes including metabolism of xenobiotics, biosynthesis of cholesterol, steroid hormones etc. Since some CYP catalyze indol oxidation to isatin, we have hypothesized that isatin can regulate protein-protein interactions (PPI) between components of the CYP system thus representing a (negative?) feedback mechanism. The aim of this study was to investigate a possible effect of isatin on interaction of human CYP with cytochrome b5 (CYB5A). Using the optical biosensor test system employing surface plasmon resonance (SPR) we have investigated interaction of immobilized CYB5A with various CYP in the absence and in the presence of isatin. The SPR-based experiments have shown that a high concentration of isatin (270 mM) increases Kd values for complexes CYB5A/CYP3А5 and CYB5A/CYP3A4 (twofold and threefold, respectively), but has no influence on complex formation between CYB5A and other CYP (including indol-metabolizing CYP2C19 and CYP2E1). Isatin injection to the optical biosensor chip with the preformed molecular complex CYB5A/CYP3A4 caused a 30%-increase in its dissociation rate. Molecular docking manipulations have shown that isatin can influence interaction of CYP3А5 or CYP3A4 with CYB5A acting at the contact region of CYB5A/CYP.

The amyloid-beta peptide 1-42 formed during proteolytic processing of the amyloid precursor protein (APP) plays a key role in the development or progression of Alzheimer's disease (AD) and other pathologies associated with formation of protein aggregates in the central nervous system. Recent proteomic profiling of mouse and rat brain preparations by means of beta-amyloid peptide immobilized on Affigel-10 revealed a large group of amyloid-binding proteins (n>80). Many (about 25%) of these proteins were previously identified as isatin-binding proteins. The aim of this study was to validate direct interaction between beta-amyloid peptide and highly purified intact and oxidized peroxiredoxin, M-type pyruvate kinase, alpha-enolase, and the effect of isatin on this interaction. The study performed using SPR-based Biacore 3000 and Biacore X100 biosensors has shown that all the proteins form molecular complexes with immobilized beta-amyloid peptide. The Kd values for these complexes varied from 8.36х10-8 M (peroxiredoxin) to 1.97х10-6 M (alpha-enolase). Oxidative modification of investigated proteins caused opposite effects on complexes of these peptides with beta-amyloid. The endogenous neuroprotector isatin increased dissociation of complexes formed by beta-amyloid peptide with both intact proteins (peroxiredoxin, glyceraldehyde-3-phosphate dehydrogenase) and/or oxidized proteins (peroxiredoxin, pyruvate kinase) used in this study.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a classical glycolytic redox sensitive enzyme, exhibits various non-glycolytic functions, which are considered to be especially important for progression of various neurodegenerative diseases. GAPDH binds isatin (indole-dione-2,3), an endogenous indole often used as a parent component in numerous derivatives demonstrating diverse pharmacological (including neuroprotector) activities. In this study we have investigated binding of intact and mildly oxidized GAPDH to immobilized isatin, using an optical biosensor technique, employing surface plasmon resonance (SPR), and the effect of isatin as a probe for this binding. Mild GAPDH oxidation by 70 mM H(2)O(2) increased enzyme dissociation from immobilized isatin. Since GAPDH is considered as a putative target for various neuroprotector agents, this suggests that its redox state determines sensitivity to neuroprotective agents, and oxidative stress typical for various neurodegenerative disorders may significantly reduce pharmacological effectiveness of such compounds.

Isatin-binding activity of mice liver proteins has been investigated in the samples from the control and flight groups by using the methods of biosensor and proteomic analysis. It was found the higher isatin-binding activity in mice of flight group. The content of a number of individual isatin-binding proteins in the samples of the flight groups differ slightly from the ground control. However, in samples from animals which have weekly post-flight adaptation, the level of certain proteins was significantly increased. The latter allows us to assume that the main events in the proteome of mice (at least in subproteome of isatin-binding proteins), occurs in early post-flight period.

Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with high affinity and specificity. Usually, they are experimentally selected using the SELEX method. Here, we describe an approach toward the in silico selection of aptamers for proteins. This approach involves three steps: finding a potential binding site, designing the recognition and structural parts of the aptamers and evaluating the experimental affinity. Using this approach, a set of 15-mer aptamers for cytochrome P450 51A1 was designed using docking and molecular dynamics simulation. An experimental evaluation of the synthesized aptamers using SPR biosensor showed that these aptamers interact with cytochrome P450 51A1 with Kd values in the range of 10(-6)-10(-7) M., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Molecular interactions between proteins redox partners (cytochromes Р450 3А4, 3А5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes Р450 3А4 and 3А5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435  -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 in the presence of its substrate testosterone.

The amyloid-β peptide is considered as a key player in the development and progression of Alzheimer's disease (AD). Although good evidence exists that amyloid-β accumulates inside cells, intracellular brain amyloid-binding proteins remain poorly characterized. Proteomic profiling of rat brain homogenates, performed in this study, resulted in identification of 89 individual intracellular amyloid-binding proteins, and approximately 25% of them were proteins that we had previously identified as specifically binding to isatin, an endogenous neuroprotector molecule. A significant proportion of the amyloid-binding proteins (more than 30%) are differentially expressed or altered/oxidatively modified in AD patients. Incubation of brain homogenates with 70 µM hydrogen peroxide significantly influenced the profile of amyloid-β binding proteins and 0.1 mM isatin decreased the number of identified amyloid-β binding proteins both in control and hydrogen peroxide treated brain homogenates. The effects of hydrogen peroxide and isatin have been confirmed in optical biosensor experiments with purified glyceraldehyde-3-phosphate dehydrogenase, one of the known crucial amyloid-β binding proteins (also identified in this study). Data obtained suggest that isatin protects crucial intracellular protein targets against amyloid binding, and possibly favors intracellular degradation of this protein via preventing formation of amyloid-β oligomers described in the literature for some isatin derivatives.

Protein ubiquitination is considered as an important mechanism that is responsible not only for specific labeling of proteins for their subsequent degradation but also for localization of proteins in the cell and regulation of protein-protein interactions. In the context of protein-protein interactions binding of (mono/poly)ubiquitinated molecules to proteins containing specific ubiquitin binding domains appear to play the decisive role. Although formation of the ubiquitin interactome has been demonstrated for cytosol, involvement of mitochondria and associated extramitochondrial proteins into such interactions still requires detailed investigation. In this study using an optical biosensor we have demonstrated binding of proteins of mouse brain mitochondrial lysates to immobilized monomeric ubiquitin. Model purified proteins, which are known to be associated with the outer mitochondrial compartment (glyceraldehyde-3-phosphate dehydorgenase, creatine phosphokinase), interacted with immobilized ubiquitin as well as with each other. This suggests that (poly)ubiquitinated chains may be involved in protein-protein interactions between ubiquitinated and non-ubiquitinated proteins and thus may contribute to formation of (mitochondrial) ubiquitin subinteractome.

The cholesterol biosynthesis regulation is the important part of the hypercholesterolemia diseases therapy. The inhibition of the post-squalene cholesterol biosynthesis steps provide the alternative to classic statin therapy. Sterol-14a-demethylase (CYP51) is one of the hypothetical targets for it. In this work the screening of the ability to interact with human CYP51 (CYP51A1) for the nature low-weight compounds with steroid-like scaffold were performed by integration of the surface plasmon resonance biosensor and spectral titration methods. The results of the selection were 4 compounds (betulafolientriol, holothurin A, teasaponin, capsicoside A) witch had high affinity to the CYP51A1 active site. These data extend the range of compounds which may be used as specific inhibitors of CYP51 and give the permission to suggest the dynamic of the enzyme.

Molecular interactions between proteins redox partners (cytochromes P450 3A4, 3A5 and cytochrome b5) within the monooxygenase system, which is known to be involved in drug biotransformation, were investigated. Human cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) form complexes with various cytochromes b5: the microsomal (b5mc) and mitochondrial (b5om) forms of this protein, as well as with 2 "chimeric" proteins, b5(om-mc), b5(mc-om). Kinetic constants and equilibrium dissociation constants were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was only observed upon their interactions with cytochrome b5om. Electroanalytical characteristics of electrodes with immobilized hemoproteins were obtained. The electrochemical analysis of CYP3A4, CYP3A5, b5mc, b5om, b5(om-mc), and b5(mc-om) immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 - -0.350 V (vs. Ag/AgCl). Cytochrome b5mc was shown to be capable of stimulating the electrocatalytic activity of CYP3A4 to testosterone.

The SPR assay for human cytochrome P450 51A1's (CYP51A1) ligand screening was developed. Assay has been validated with known azole inhibitors of cytochrome P450s. The studied azoles selectively interacted with human cytochrome P450 51A1, which showed the highest affinity towards ketoconazole. The efficiency of the SPR assay was showed with 19 steroid and triterpene compounds, which were not investigated as potential ligands of CYP51A1.

A highly sensitive reverse sandwich immunoassay for the detection of human cardiac myoglobin (cMb) in serum was designed utilizing a gold nanoparticle (AuNP)-enhanced surface plasmon resonance (SPR) biosensor. First, a monoclonal anti-cMb antibody (Mab1) was covalently immobilized on the sensor surface. AuNPs were covalently conjugated to the second monoclonal anti-cMb antibody (Mab2) to form an immuno-gold reagent (Mab2-AuNP). The reverse sandwich immunoassay consists of two steps: (1) mixing the serum sample with Mab2-AuNP and incubation for the formation of cMb/Mab2-AuNP complexes and (2) sample injection over the sensor surface and evaluation of the Mab1/cMb/Mab2-AuNP complex formation, with the subsequent calculation of the cMb concentration in the serum. The biosensor signal was amplified approximately 30-fold compared with the direct reaction of cMb with Mab1 on the sensor surface. The limit of detection of cMb in a human blood serum sample was found to be as low as 10 pM (approx. 0.18 ng mL(-1)), and the inter-assay coefficient of variation was less than 3%. Thus, the developed SPR-based reverse sandwich immunoassay has a sensitivity that is sufficient to measure cMb across a wide range of normal and pathological concentrations, allowing an adequate estimation of the disease severity and the monitoring of treatment., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Here, we describe the analysis of kinetic and thermodynamic parameters for binding of peptide and nonpeptide dimerization inhibitors to immobilized HIV protease (HIVp) monomers by using surface plasmon resonance. Molecular interactions were investigated at different inhibitors concentrations (0-80 microM) and temperatures (15-35 degrees C). The kinetic, equilibrium and thermodynamic parameters have been determined. It was found that both inhibitors were characterized by similar interaction parameters. The complex formation is entropically driven process for both inhibitors. The entropic term(-TdeltaS) had the value about -20 kcal/mol while the enthalpic term (deltaH) had the positive value about 14 kcal/mol and counteracted the complex formation.

NADPH-cytochrome P450 reductase (CPR) transfers two reducing equivalents derived from NADPH via FAD and FMN to microsomal P450 monooxygenases in one-electron transfer steps. The crystal structure of yeast CPR (yCPR) contains a surface-exposed FMN binding site (FMN2 site) at the interface of the FMN binding and connecting domains, in addition to the single buried site that has been observed in rat CPR. This finding provides a testable hypothesis of how intramolecular (between FAD and FMN) and intermolecular (between FMN and P450) electron transfer may occur in CPR. To verify that occupancy of the FMN2 site is not an artifact of crystallization, a surface plasmon resonance (SPR) biosensor technique has been applied to probe the selectivity of this site under functional conditions. A series of kinetic and equilibrium binding experiments involving yCPR immobilized on different sensor chip surfaces was performed using FMN and FAD, as well as FMN-derived compounds, including riboflavin, dimethylalloxazine, and alloxazine, and other molecules that resemble the planar isoalloxazine ring structure. Only FMN and FAD showed stoichiometric binding responses. Binding affinity for FMN was in the submicromolar range, 30 times higher than that for FAD. Association kinetic rates for the yCPR/FMN complex were up to 60-fold higher than for the yCPR/FAD complex. Taken together, these data indicate that (i) the surface-exposed site in yCPR is highly selective toward binding flavins, (ii) binding of FMN in this site is notably favored, and finally, (iii) both the phosphate group and the isoalloxazine ring of FMN are essential for binding.

Aptamers interacting selectively with the anion-binding exosites 1 and 2 of thrombin were merged into dimeric oligonucleotide constructs with use of a poly-(dT)-linker of 35 nucleotides (nt) long. Complexes of thrombin with the aptamers and their hetero- and homodimeric constructs were measured using the optical biosensor Biacore-3000. K(D) values measured for the hetero- and homodimeric constructs were correspondingly 25-30- and 2-3-fold lower than those for the primary aptamers. Analysis of temperature dependencies of K(D) values within the temperature interval of 10 degrees C-40 degrees C has shown that the values of enthalpy change deltaH upon formation of complexes of thrombin with the aptamers and the hetrodimeric construct are close. The value of the entropy change deltaS upon complex formation of thrombin with the aptamer heterodimeric construct was 1.5-2-fold higher than deltaS values for the complexes with the aptamers. The complex formation and dissociation rates increased with the elevation of temperature from 10 degrees C to 37 degrees C. However, the dissociation rate for the complex of thrombin with the heterodimeric construct was evidently lower that that for the complexes with the aptamers.

Using two DNA aptamers selectively recognizing anion-binding exosites 1 and 2 of thrombin as a model, it has been demonstrated that their conjugation by a poly-(dT)-linker (ranging from 5 to 65 nt in length) to produce aptamer heterodimeric constructs results into affinity enhancement. The apparent dissociation constant (Kd(app)) measured at the optical biosensor Biacore-3000 for complexes of thrombin with the heterodimeric constructs reached minimum values (Kd(app) = 0.2-0.4 nM) which were approximately 30-fold less than for the complexes with the primary aptamers. A photoaptamer heterodimeric construct was designed connecting photoaptamer and aptamer sequences with the poly-(dT)-linker of 35 nt long. The photoaptamer used could form photo-induced cross-links with the exosite 2 of thrombin and the aptamer used could bind to the exosite 1. The measured value of Kd(app) for the photoaptamer construct was approximately 40-fold less than that for the primary photoaptamer (5.3 and 190 nM, respectively). Upon exposure to the UV radiation at 308 nm of the equimolar mixtures of thrombin with the photoaptamer construct, the equal yield of the crosslinked complexes was observed at concentrations which were lower by two orders of magnitude than in the case of the primary photoaptamer. It was found that concurrently with crosslinking to thrombin a photo-induced inactivation of the photoaptamer occurs presumably due to formation of the intermolecular crosslinking.

Using an optical biosensor Biacore 3000 the interaction of human recombinant cytokeratins (CK) with isatin analogues (5-aminocaproyl-isatin and 5-aminoisatin) immobilized on the CM-5 chip has been investigated. CK-14 effectively interacted with 5-aminocaproyl-isatin immobilized on the carboxymethyl dextran chip surface, but not with a "shorter" analogue (5-aminoisatin). In contrast to CK14 CK8 effectively interacted only with 5-aminoisatin. In both cases cytokeratin binding with the immobilized isatin analogues was characterized by rather high affinity (Kd of 0.7 microM for the pair CK14/immobilized 5-aminocaproylisatin and 1.7 microM for the pair CK8/immobilized 5-aminoisatin). CK20 did not interact with both immobilized isatin analogues. Taking into consideration non-specific binding of mouse CK14 and rat CK8 with 5-aminocaproyl-Sepharose we have performed comparative analysis of amino acid sequences of human, mouse, and rat CK8 and CK14. The data obtained suggest that in the case of human, mouse, and rat CK14 the N-terminal domain is the most variable among these species, whereas the major differences between amino acid sequences of human, mouse, and rat CK8 have been found both in N-terminal and C-terminal regions.

Currently inhibitors of protein-protein interactions are considered as perspective prototypes of new generation of drugs. The most attractive targets for such inhibitors are the oligomeric enzymes which active sites are formed by amino acid residues from different subunits. The classic example of such enzyme is HIV protease (HIVp), active only in the homodimeric form. We have developed a new approach for experimental screening of HIVp dimerization inhibitors. It is based on the original biosensoric test-system for differential analysis of interaction of tested substances with HIVp dimers and monomers. Using this test-system we have analyzed the most perspective candidate substances predicted by method of virtual screening, and some derivatives of glycyrrhizin, triterpenic and steroid glycosides. As a result we found one compound, which mainly interacts with HIVp monomers and inhibits in vitro activity of this enzyme with IC50 of about 10(-6) M.

Hybridization of telomeric repeats with a complementary oligonucleotide probe was studied by the surface plasmon resonance method. Conjugation of the probe with streptavidin and gold nanoparticles was shown to amplify the signal at similar concentrations of this probe (by 60 and 300 times, respectively). Nanoparticles can be used for biosensor signal amplification in studying the telomerase activity of malignant cells.

Protein-protein and protein-ligand interactions play a central role in biochemical reactions, and understanding these processes is an important task in different fields of biomedical science and drug discovery. Proteins often work in complex assemblies of several macromolecules and small ligands. The structural and functional description of protein-protein interactions (PPI) is very important for basic-, as well as applied research. The interface areas of protein complexes have unique structure and properties, so PPI represent prospective targets for a new generation of drugs. One of the key targets of PPI inhibitors are oligomeric enzymes. This report shows interactive links between virtual and experimental approaches in a total pipeline "from gene to drug" and using Surface Plasmon Resonance technology for experimentally assessing PPI. Our research is conducted on two oligomeric enzymes -- HIV-1 protease (HIVp) (homo-dimer) and bacterial L-asparaginase (homo-tetramer). Using methods of molecular modeling and computational alanine scanning we obtained structural and functional description of PPI in these two enzymes. We also presented a real example of application of integral approach in searching inhibitors of HIVp dimerization -- from virtual database mining up to experimental testing of lead compounds.

The interaction of inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated. The following enzymes were tested: serine proteases (trypsin, alpha-chymotrypsin, plasmin, thrombin, kallikrein), cysteine protease (papain) and aspartic protease (pepsin). Inhibitor VJ interacted only with trypsin and 6-chymotrypsin. Kinetic and thermodynamics parameters of intermolecular complexes formation were determined: KD = 7,38 x 10(-8) M and 9,93 x 10(-7) M for pairs InhVJ/trypsin and InhVJ/alpha-chymotrypsin, respectively.

The glycolytic enzyme, pyruvate kinase, exhibits moderate affinity [3H]isatin binding (KD approximately 10 microM), which is inhibited by ATP (IC50 25 microM) and deprenyl (IC50 5 microM). Interaction of pyruvate kinase with isatin and its inhibition by ATP and deprenyl has also been confirmed using an independent biosensor technique and immobilized isatin analogue, aminoisatin. This effect has some specificity because the enzyme, creatine phosphokinase, does not exhibit specific isatin-binding. It is suggested that interaction of pyruvate kinase with isatin may reflect some non-glycolytic functions of this enzyme.

Bacterial L-asparaginases catalyzing hydrolysis of L-asparagine up to L-aspartate and ammonia, are used in medical practice for treatment of acute lymphoblastic leukemia. The long-term therapy with these preparations is accompanied by a number of side effects, which are attributed to glutaminase activity of L-asparaginase. Substrate specificity and activity of L-asparaginases are directly associated with the process of enzyme oligomerization. It is active only in the tetrameric form as the active sites are located in contact areas between monomers. The present work is devoted to homology modeling of spatial structure of L-asparaginase from Erwinia carotovora, the comparative molecular-graphic analysis of subunits interfaces, as well as development of experimental approach for enzyme oligomerization study. L-asparaginase was immobilized on a CM5 chip surface of optical biosensor Biacore 3000 based on the surface plasmon resonance technology. The dissociation process of enzyme tetrameric complexes up to monomers and subsequent oligomerization process have been registered.

Isatin is an endogenous indole widely distributed in mammalian tissues and body fluids. The presence of isatin-binding proteins has been recognised in particulate and soluble fractions of various organs and tissues. However, identified targets of isatin action (monoamine oxidase, natriuretic peptide receptor type A and soluble NO-stimulated guanylate cyclase) cannot account for all biological activity of this compound. Highly purified glycerol-3-phosphate dehydrogenase (GPDH) from rabbit muscle effectively interacts with the isatin analogue immobilised on the cuvette of IAsys optical biosensor. This effect was specific because the other NAD-dependent cytosolic enzyme purified from rabbit muscle, lactate dehydrogenase failed to interact with the immobilised isatin analogue. Replacement of the cuvette medium for washing buffer did not cause total dissociation of GPDH-isatin complexes. This suggests involvement of several types of enzyme-isatin interaction including tight binding. Low (10 microM) and high (100 microM) concentrations of isatin caused different effects on GPDH activity: the former significantly increased apparent Km for NAD, whereas the latter decreased apparent Vmax and increased Km.

The detection of hepatitis B virus surface antigen (HBsAg) with the use of a model IAsys+ two-channel optical biosensor is based on the registration of interaction between anti-HBs monoclonal antibodies forming the surface layer of the biochip of the biosensor cuvette and blood serum HBsAg. For the first time a two-channel optical biosensor has been used for the detection of HBsAg in blood serum samples. The comparative analysis of the detection of HBsAg by two methods, viz. with the use of an optical biosensor and the enzyme immunoassay, has demonstrated lower sensitivity, but higher specificity of the detection of this antigen by means of a model IAsys+ biosensor with the biochip, prepared in the process of the work. The main advantages of the biosensor detection lie in the registration of interaction in real time without introducing special markers into the molecules under study.

The interaction between two different monoclonal antibodies (Mabs) and their corresponding bispecific antibodies (Babs) with immobilized antigens was investigated using an optical biosensor (IAsys). The analyzed panel of affinity-purified antibodies included two parental Mabs (one of which was specific to human IgG (hIgG), and another one to horseradish peroxidase (HRP)), as well as Babs derived thereof (anti-hIgG/HRP). Babs resulting from the fusion of parental hybridomas bear two antigen-binding sites toward two different antigens and thus may interact with immobilized antigen through only one antigen-binding site (monovalently). Using an IAsys biosensor this study shows that the bivalent binding of Mabs predominates over the monovalent binding with immobilized HRP, whereas anti-hIgG parental Mabs were bound monovalently to the immobilized hIgG. The observed equilibrium association constant (K(ass)) values obtained in our last work [1] by solid-phase radioimmunoassay are consistent with those constants obtained by IAsys. The K(ass) of anti-HRP Mabs was about 50 times higher than that of anti-HRP shoulder of Babs. The dissociation rate constant (k(diss)) for anti-HRP shoulder of Babs was 21 times higher than k(diss) for anti-HRP Mabs. The comparison of the kinetic parameters for bivalent anti-HRP Mabs and Babs derived from anti-Mb/HRP and anti-hIgG/HRP, allowed to calculate that 95% of bound anti-HRP Mabs are bivalently linked with immobilized HRP, whereas only 5% of bound anti-HRP Mabs are monovalently linked. In general, the data obtained indicate that Babs bearing an enzyme-binding site may not be efficiently used instead of traditional antibody-enzyme conjugates in the case of binding of bivalent Mabs.

The interaction between two monoclonal antibodies (mAbs) and their corresponding bispecific antibody (bAb) with immobilized antigens has been examined using a resonant mirror biosensor (IAsys). BAbs were produced by cell fusion. The analysed panel of affinity-purified antibodies included two parental mAbs, one specific to human IgG (hIgG), and another specific to horseradish peroxidase (HRP), and a bAb derived thereof (anti-hIgG/HRP). The real-time analysis showed the drastic differences in the avidity of bivalent anti-HRP mAbs and anti-HRP shoulder of bAbs. Thus, the observed equilibrium association constant (K(ass)) of anti-HRP mAbs was about 50 times higher that of anti-HRP shoulder of bAbs. The ratio of association rate constants (k(ass)) of mAbs and bAbs was about two, due to the statistical factor of two binding sites per bivalent antibody molecule. However, the dissociation rate constant (k(diss)) of anti-HRP shoulder of bAbs was 21 times higher k(diss) of anti-HRP mAbs. The comparison with the theoretical model shows that these observations are consistent only with a situation in which bivalent binding of mAbs with immobilized HRP predominates over monovalent binding. On the contrary, the second parental mAb (anti-hIgG) did not show the increase in avidity due to bivalent binding, compared to the anti-hIgG shoulder of bAbs, suggesting that this mAb was bound monovalently to immobilized hIgG. The K(ass) values determined by solid-phase radioimmunoassay (RIA) yielded figures almost overlapping with those obtained by IAsys. The results of the comparison of bAbs and mAbs are discussed from the viewpoint of the use of bAbs in heterogeneous systems. On the other hand, these data demonstrate that real-time analysis of antibody binding parameters in IAsys biosensor is valuable for the selection of mAbs and bAbs with desired features, for different fields of application.

An original method for the integral evaluation of tissue and subcellular distribution of isatin binding proteins has been developed. This method is based on continuous monitoring of changes of optical characteristics that accompany complex formation between a ligand (immobilized on dextran bed of IAsys biosensor cell) and its soluble receptor. Solubilisation of tissue preparations and subcellular fractions with detergent (1% Triton X-100) is the important preconditions for the applicability of this method. The immobilisation of 5-aminoisatin was achieved by peptide bond formation between amino group of this isatin analogue and carboxyl group of the dextran bed of the biosensor cell. Addition of Triton X-100 treated preparations of membrane and soluble fractions of rat brain, liver, heart, and kidneys to the biosensor cell resulted in appearance of the characteristic response, indicating complex formation with the immobilised isatin analogue. The magnitude and a shape of kinetic curve vary in these samples. Isatin binding proteins predominated in membrane fractions of brain, liver and heart preparations whereas in the kidneys the highest isatin-binding response was detected in the soluble fraction. The distribution of isatin binding sites in the particulate fraction reduced in the following order: brainstem > brain hemispheres = cerebellum > heart > kidneys > liver. In the soluble fraction there was different rank of isatin binding activity: kidneys > heart > brainstem = brain hemispheres > liver > cerebellum. Liver outer mitochondrial membranes are characterised by the higher isatin-binding than mitochondria. Treatment of mitochondria with clorgyline and deprenyl, specifically inhibiting MAO A and B, respectively, significantly reduced the magnitude of the biosensor response and changed the shape of the kinetic curve. These data are consistent with the notion that within mitochondria MAOs are the major targets of isatin.

A new method for detection of hepatitis B surface antigen (HBsAg) has been developed. It employees IAsys optical biosensor registration kinetics of HBsAg complexes with monoclonal antibodies. Detection of intermolecular interactions is accompanied by changes of the light refraction coefficient in the sensitive layer of the biosensor cuvette. The main advantage of this diagnostic technique consists in rapid registration of these interactions in real time, without any introduction of special labels into analysing molecules. The optical biosensor method was successfully employed for the detection of HBsAg in human blood serum. A comparative study of HBsAg detection by the optical biosensor and by immunoenzyme analysis demonstrated high specificity of HBsAg detection by this new method.

A phospholipid-containing biochip was created by covalently immobilizing phospholipids on the optical biosensor's aminosilane cuvette and employed to monitor the interactions of the membrane and water-soluble proteins in cytochrome P450-containing monooxygenase systems with planary layers of dilauroylphosphatidylethanolamine (DLPE) and distearoylphosphatidylethanolamine (DSPE), differing in acyl chain length. It was shown that the full-length membrane proteins-cytochrome P4502B4 (d-2B4), cytochrome b5 (d-b5) and NADPH-cytochrome P450 reductase (d-Fp)-readily incorporated into the phospholipids. The incorporation was largely due to hydrophobic interactions of membranous protein fragments with the phospholipid layer. However, electrostatic forces were also but not always involved in the incorporation process. They promoted d-Fp incorporation but had no effect on d-b5 incorporation. In low ionic strength buffer, no incorporation of these two proteins into the DSPE lipid layer was observable. Incorporation of d-b5 into the DLPE layer was abruptly increased at temperatures exceeding phospholipid phase transition point. Incorporation of d-2B4 was dependent on its aggregation state and decreased with increasing protein aggregability. Water-soluble proteins either would not interact with the phospholipid layer (adrenodoxin) or would bind to the layer at the cost of only electrostatic (albumin) or both electrostatic and hydrophobic (P450cam) interactions., (Copyright 2001 John Wiley & Sons, Ltd.)

The kinetics of hybridization of 11-meric and 14-meric oligonucleotides, dTGGGAAGAGGG (ODN-11) and dTGGGAAGAGG GTCA (ODN-14), with 14-meric oligonucleotide dpTGACCCTCT TCCCA (p14) attached to the surface of a cuvette was studied by the resonant mirror method. The treatment of the experimental curves with exponential equations leads to the following values for association (kas) and dissociation (kdis) rate constants at 25 degrees C: kas = 219 +/- 39 and 183 +/- 162 M-1 s-1, kdis = (2.0 +/- 0.4) x 10(-3) and (4 +/- 1) x 10(-4) s-1 for the duplexes (p14) x (ODN-11) and p14 x (ODN-14), respectively. The oligonucleotide dTGCCTTGAATGGGAA GAGGGTCA (ODN-23), which forms a hairpin structure, does not associate with p14. The data were compared with the results of melting curve detection and temperature-jump experiments. The association rate constants for ODN-11 and ODN-14 are much slower than those values in homogeneous aqueous solution. The dissociation rate constants have the same magnitude values as estimated by using association constants measured from melting curves but differ from the values estimated in temperature-jump experiments.