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. Radiochemical assay in combination with molecular docking reveals the formation of the complexes, in which proline residues turned to the interface between water and hydrophobic medium

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. Thus, we obtained the following selectivity series of calcium, magnesium, zinc, manganese and copper cations: Zn2+ > Mn2+ > Mg2+ > Ca2+ >> Cu2+. Remarkably, Cu2+ appeared to suppress the UA-mediated proton transport in both lipid membrane systems. The data on the divalent metal cation/proton exchange were supported by circular dichroism spectroscopy of UA in the presence of the corresponding cations

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. The effect of the peptides on the activity of CcO was studied in solution and in membranes. The secondary structure of the peptides was examined by molecular dynamics, circular dichroism spectroscopy, and testing the ability to form membrane pores. P4 was found to suppress the oxidase but not the peroxidase activity of solubilized CcO. The Ki(app) is linearly dependent on the dodecyl-maltoside (DM) concentration, indicating that DM and P4 compete in a 1:1 ratio. The true Ki is 3 μM. The deoxycholate-induced increase in Ki(app) points to a competition between P4 and deoxycholate. A1 and A4 inhibit solubilized CcO with Ki(app)~20 μM at 1 mM DM. A2 and A3 hardly inhibit CcO either in solution or in membranes. The mitochondrial membrane-bound CcO retains sensitivity to P4 and A4 but acquires resistance to A1. We associate the inhibitory effect of P4 with its binding to BABS and dysfunction of the proton channel K. Trp residue is critical for inhibition. The resistance of the membrane-bound enzyme to inhibition may be due to the disordered secondary structure of the inhibitory peptide

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. This research is focused on establishing a thermodynamic background and the conformational heterogeneity of aptamers taking a series of thrombin DNA aptamers having G-quadruplex and duplex modules as an example. A series of aptamers with similar modular structures was characterized with spectroscopic and chromatographic techniques, providing examples of the conformational homogeneity of aptamers with high inhibitory activity, as well as a mixture of monomeric and oligomeric species for aptamers with low inhibitory activity. Thermodynamic parameters for aptamer unfolding were calculated, and their correlation with aptamer functional activity was found. Detailed analysis of thrombin complexes with G-quadruplex aptamers bound to exosite I revealed the similarity of the interfaces of aptamers with drastically different affinities to thrombin. It could be suggested that there are some events during complex formation that have a larger impact on the affinity than the states of initial and final macromolecules. Possible mechanisms of the complex formation and a role of the duplex module in the association process are discussed

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

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. To determine the nature of these changes, we performed ab initio modeling, including the multiphase procedure, with the geometric bodies (helices) and restoration of the PVA structure in solution using available high-resolution structures of the homologous CP from the PVY potyvirus, based on the SAXS data. As a result, for the first time, a low-resolution structure of the filamentous PVA virus, both intact and partially degraded, was elucidated under conditions close to natural. The far-UV circular dichroism spectra of the PVA and PVAΔ32 samples differed significantly in the amplitude and position of the main negative maximum. The extent of thermal denaturation of these samples in the temperature range of 20-55°C was also different. The data of transmission electron microscopy showed that the PVAΔ32 virions were mostly rod-shaped, in contrast to the flexible filamentous particles typical of the intact virus, which correlated well with the SAXS results. In general, structural analysis indicates an importance of the CP N-terminal domain for the vital functions of PVA, which can be used to develop a strategy for combating this plant pathogen

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. These hemagglutinins have homologous tertiary structures but moderate-to-low amino acid sequence identities. Here, the experiment was inverted, targeting the same protein using a set of related, parallel G-quadruplexes. The 5′- and 3′-flanking sequences of RHA0385 were truncated to yield parallel G-quadruplex with three propeller loops that were 7, 1, and 1 nucleotides in length. Next, a set of minimal, parallel G-quadruplexes with three single-nucleotide loops was tested. These G-quadruplexes were characterized both structurally and functionally. All parallel G-quadruplexes had affinities for both recombinant hemagglutinin and influenza virions. In summary, the parallel G-quadruplex represents a minimal core structure with functional activity that binds influenza A hemagglutinin. The flanking sequences and loops represent additional features that can be used to modulate the affinity. Thus, the RHA0385–hemagglutinin complex serves as an excellent example of the hypothesis of a core structure that is decorated with additional recognizing elements capable of improving the binding properties of the aptamer