The Increase in the Peroxidase Activity of the Cytochrome with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands.

Cytochrome (CytC), a one-electron carrier, transfers electrons from complex to cytochrome oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation.

The fully reduced terminal oxidase bd-I isolated from Escherichia coli binds cyanide.

Cytochrome bd-I from Escherichia coli belongs to the superfamily of prokaryotic bd-type oxygen reductases. It contains three hemes, b, b and d, and couples oxidation of quinol by dioxygen with the generation of a proton-motive force. The enzyme exhibits resistance to various stressors and is considered as a target protein for next-generation antimicrobials.

Lysozyme binding with amikacin and levofloxacin studied by tritium probe, fluorescence spectroscopy and molecular docking.

Lysozyme complexes with amikacin and levofloxacin were studied by spectroscopy approaches as well as using a tritium probe. Tritium was used as a labeling agent to trace labeled compound concentration in a system of two immiscible liquids and in the atomic form to determine the possible position of the binding site. Co-adsorption of protein and drug at the liquid-liquid interface was analyzed by scintillation phase method that allowed us to directly determine the amount of protein and drug in the mixed adsorption layer.

Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties.

Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO.

Effect of the Coat Protein N-Terminal Domain Structure on the Structure and Physicochemical Properties of Virions of Potato Virus X and Alternanthera Mosaic Virus.

The amino acid sequences of the coat proteins (CPs) of the potexviruses potato virus X (PVX) and alternanthera mosaic virus (AltMV) share ~40% identity. The N-terminal domains of these proteins differ in the amino acid sequence and the presence of the N-terminal fragment of 28 residues (ΔN peptide) in the PVX CP. Here, we determined the effect of the N-terminal domain on the structure and physicochemical properties of PVX and AltMV virions.

G-Quadruplex Is Recognized and Preferentially Bound but Not Processed by the MutL Endonuclease from Mismatch Repair Pathway.

The human pathogen uses a homologous recombination to undergo antigenic variation and avoid an immune response. The surface protein pilin (PilE) is one of the targets for antigenic variation that can be regulated by mismatch repair (MMR) and a G-quadruplex (G4) located upstream of the promoter. Using bioinformatics tools, we found a correlation between variability and deletion of DNA regions encoding ngMutS or ngMutL proteins, the main participants in methyl-independent MMR.

Interaction of Amphipathic Peptide from Influenza Virus M1 Protein with Mitochondrial Cytochrome Oxidase.

The Bile Acid Binding Site (BABS) of cytochrome oxidase (CcO) binds numerous amphipathic ligands. To determine which of the BABS-lining residues are critical for interaction, we used the peptide P4 and its derivatives A1-A4. P4 is composed of two flexibly bound modified α-helices from the M1 protein of the influenza virus, each containing a cholesterol-recognizing CRAC motif.

Usnic Acid-Mediated Exchange of Protons for Divalent Metal Cations across Lipid Membranes: Relevance to Mitochondrial Uncoupling.

Usnic acid (UA), a unique lichen metabolite, is a protonophoric uncoupler of oxidative phosphorylation, widely known as a weight-loss dietary supplement. In contrast to conventional proton-shuttling mitochondrial uncouplers, UA was found to carry protons across lipid membranes via the induction of an electrogenic proton exchange for calcium or magnesium cations. Here, we evaluated the ability of various divalent metal cations to stimulate a proton transport through both planar and vesicular bilayer lipid membranes by measuring the transmembrane electrical current and fluorescence-detected pH gradient dissipation in pyranine-loaded liposomes, respectively.

Individual heme a and heme a contributions to the Soret absorption spectrum of the reduced bovine cytochrome c oxidase.

Bovine cytochrome c oxidase (CcO) contains two hemes, a and a, chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa-type cytochrome c oxidase consists of overlapping bands of the hemes a and a. It shows a peak at ∼444 nm and a distinct shoulder at ∼425 nm.

Equilibrium Between Dimeric and Monomeric Forms of Human Epidermal Growth Factor is Shifted Towards Dimers in a Solution.

An interplay between monomeric and dimeric forms of human epidermal growth factor (EGF) affecting its interaction with EGF receptor (EGFR) is poorly understood. While EGF dimeric structure was resolved at pH 8.1, the possibility of EGF dimerization under physiological conditions is still unclear.

Thermal remodelling of Alternanthera mosaic virus virions and virus-like particles into protein spherical particles.

The present work addresses the thermal remodelling of flexible plant viruses with a helical structure and virus-like particles (VLPs). Here, for the first time, the possibility of filamentous Alternanthera mosaic virus (AltMV) virions' thermal transition into structurally modified spherical particles (SP) has been demonstrated. The work has established differences in formation conditions of SP from virions (SPV) and VLPs (SPVLP) that are in accordance with structural data (on AltMV virions and VLPs).

The Structure of the Potato Virus A Particles Elucidated by Small Angle X-Ray Scattering and Complementary Techniques.

Potato virus A (PVA) protein coat contains on its surface partially unstructured N-terminal domain of the viral coat protein (CP), whose structural and functional characteristics are important for understanding the mechanism of plant infection with this virus. In this work, we investigated the properties and the structure of intact PVA and partially trypsinized PVAΔ32 virions using small-angle X-ray scattering (SAXS) and complimentary methods. It was shown that after the removal of 32 N-terminal amino acids of the CP, the virion did not disintegrate and remained compact, but the helical pitch of the CP packing changed.

Lysozyme-dalargin self-organization at the aqueous-air and liquid-liquid interfaces.

An experimental study of protein-peptide binding was performed by means of radiochemical and spectroscopic methods. Lysozyme and dalargin were chosen due to their biological and physiological importance. By means of tensiometry and radiochemical assays, it was found that dalargin possesses rather high surface activity at the aqueous-air and aqueous-p-xylene interfaces to be substituted by protein.

The Functional Role of Loops and Flanking Sequences of G-Quadruplex Aptamer to the Hemagglutinin of Influenza a Virus.

Nucleic acid aptamers are generally accepted as promising elements for the specific and high-affinity binding of various biomolecules. It has been shown for a number of aptamers that the complexes with several related proteins may possess a similar affinity. An outstanding example is the G-quadruplex DNA aptamer RHA0385, which binds to the hemagglutinins of various influenza A virus strains.

The cytoplasmic tail of influenza A/H1N1 virus hemagglutinin is β-structural.

Influenza A/H1N1 virus hemagglutinin (HA) is an integral type I glycoprotein that contains a large glycosylated ectodomain, a transmembrane domain, and a cytoplasmic tail (CT) of 10-14 amino acid residues. There are absolutely no data on the secondary or tertiary structure of the HA CT, which is important for virus pathogenesis. Three highly conserved cysteines are post-translationally modified by the attachment of fatty acid residues that pin the CT to the lipid membrane inside the virion.

Structural peculiarities of lysozyme - PLURONIC complexes at the aqueous-air and liquid-liquid interfaces and in the bulk of aqueous solution.

Interaction between proteins and synthetic polymers that represent a perspective potential in drug delivery or/and already used in medicine plays a key role in biological functioning of both molecules along with a system as a whole. In present study association between hen egg white lysozyme and Pluronic triblock-copolymers (L121, P123 and F127) in the bulk of the solution as well as at the aqueous-air and liquid-liquid interfaces was analyzed by means of spectroscopic and radiochemical assay. In protein-Pluronic complexes lysozyme keeps the secondary structure (CD and SAXS data results), while fluorescence and UV-analysis indicates changes in the local surrounding of fluorophoric amino acid residues.

Structural and Functional Aspects of G-Quadruplex Aptamers Which Bind a Broad Range of Influenza A Viruses.

An aptamer is a synthetic oligonucleotide with a unique spatial structure that provides specific binding to a target. To date, several aptamers to hemagglutinin of the influenza A virus have been described, which vary in affinity and strain specificity. Among them, the DNA aptamer RHA0385 is able to recognize influenza hemagglutinins with highly variable sequences.

Putative Mechanisms Underlying High Inhibitory Activities of Bimodular DNA Aptamers to Thrombin.

Nucleic acid aptamers are prospective molecular recognizing elements. Similar to antibodies, aptamers are capable of providing specific recognition due to their spatial structure. However, the apparent simplicity of oligonucleotide folding is often elusive, as there is a balance between several conformations and, in some cases, oligomeric structures.

The alpha helix 1 from the first conserved region of HIV1 gp120 is reconstructed in the short NQ21 peptide.

Investigations of short peptides that can be used in the next phase of synthetic HIV1 vaccine development are an urgent goal, as well as investigations of peptides that can be used in immunological tests with the aim to check the titer of antibodies against the alpha helix 1 from the first conserved region of HIV1 gp120 that are known to cause antibody-dependent cellular cytotoxicity (ADCC). The aim of this work was to study the structure of the NQ21 peptide corresponding to the less mutable part of the first conserved region of HIV1 gp120 (residues 94-114). The NQ21 peptide and its conjugate with biotin (biotin-NQ21) are absolutely alpha-helical in phosphate buffer solutions at pH = 6.

Engineering a carotenoid-binding site in Dokdonia sp. PRO95 Na-translocating rhodopsin by a single amino acid substitution.

Light-driven H, Cl and Na rhodopsin pumps all use a covalently bound retinal molecule to capture light energy. Some H-pumping rhodopsins (xanthorhodopsins; XRs) additionally contain a carotenoid antenna for light absorption. Comparison of the available primary and tertiary structures of rhodopsins pinpointed a single Thr residue (Thr216) that presumably prevents carotenoid binding to Na-pumping rhodopsins (NaRs).

Menthols as Chiral Auxiliaries for Asymmetric Cycloadditive Oligomerization: Syntheses and Studies of β-Proline Hexamers.

To produce a novel class of structurally ordered poly-β-prolines, an emergent method for synthesizing chiral β-peptide molecular frameworks was developed based on 1,3-dipolar cycloaddition chemistry of azomethine ylides. Functionalized short β-peptides with up to six monomeric residues were efficiently synthesized in homochiral forms using a cycloadditive oligomerization approach. X-ray, NMR, and CD structural analyses of the novel β-peptides revealed secondary structure features that were generated primarily by Z/E-β-peptide bond isomerism.

Heating-induced transition of Potyvirus Potato Virus A coat protein into β-structure.

In our previous communication, we have reported that virions of plant Potyvirus Potato Virus A (PVA) have a peculiar structure characterized by high content of disordered regions in intravirus coat protein (CP). In this report, we describe unusual properties of the PVA CP. With the help of a number of physicochemical methods, we have observed that the PVA CP just released from the virions by heating at 60-70 °C undergoes association into oligomers and transition to β- (and even cross-β-) conformation.

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.

Optical and magneto-optical activity of cytochrome bd from Geobacillus thermodenitrificans.

Cytochromes bd are terminal oxidases in the respiratory chains of many prokaryotic organisms. They reduce O₂ to 2H₂O at the expense of electrons extracted from quinol. The oxidases can be divided into two subfamilies, L and S, based on the presence of either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I designated as 'Q-loop'.

Circular dichroism spectra of cytochrome c oxidase.

Circular dichroism spectra of bovine heart aa(3)-type cytochrome c oxidase have been studied with a major focus on the Soret band π → π* transitions, B(0(x,y)), in the two iron porphyrin groups of the enzyme. The spectra of the fully reduced and fully oxidized enzyme as well as of its carbon monoxide and cyanide complexes have been explored. In addition, CD spectra of the reduced and oxidized ba(3)-type cytochrome c oxidase from Thermus thermophilus were recorded for comparison.