Characterization Of Such Interactions Research Articles

Genetically Engineered MRI-Trackable Extracellular Vesicles as SARS-CoV-2 Mimetics for Mapping ACE2 Binding In Vivo.

The elucidation of viral-receptor interactions and an understanding of virus-spreading mechanisms are of great importance, particularly in the era of a pandemic. Indeed, advances in computational chemistry, synthetic biology, and protein engineering have allowed precise prediction and characterization of such interactions. Nevertheless, the hazards of the infectiousness of viruses, their rapid mutagenesis, and the need to study viral-receptor interactions in a complex in vivo setup call for further developments. Here, we show the development of biocompatible genetically engineered extracellular vesicles (EVs) that display the receptor binding domain (RBD) of SARS-CoV-2 on their surface as coronavirus mimetics (EVsRBD). Loading EVsRBD with iron oxide nanoparticles makes them MRI-visible and, thus, allows mapping of the binding of RBD to ACE2 receptors noninvasively in live subjects. Moreover, we show that EVsRBD can be modified to display mutants of the RBD of SARS-CoV-2, allowing rapid screening of currently raised or predicted variants of the virus. The proposed platform thus shows relevance and cruciality in the examination of quickly evolving pathogenic viruses in an adjustable, fast, and safe manner. Relying on MRI for visualization, the presented approach could be considered in the future to map ligand-receptor binding events in deep tissues, which are not accessible to luminescence-based imaging.

State-Dependent and Mutation-Induced Differences in Protein-Lipid Interactions in the Na,K Atpase

Specific protein-lipid interactions have emerged as allosteric regulators of membrane protein function. However, the intrinsic dynamic nature of both the protein and the surrounding membrane render structural and biophysical characterization of such interactions are extremely challenging. The sodium-potassium transporter (Na,K ATPase) helps maintain critical electrochemical gradients by an ATP-dependent switch of three intracellular Na+ ions and two extracellular K+ ions across the membrane. Recent experimental findings have pinpointed a few locations of protein-lipid interactions of functional importance. However, a detailed description of how lipid interactions change during the Na,K ATPase membrane switch, in particular in disease mutant forms of the proteins, is currently lacking. Here, we used all-atom molecular dynamics simulations to characterize interactions by annular lipids to sodium- and potassium-bound states of Na,K ATPase in an asymmetric, multicomponent mimic of the plasma membrane. We specifically identified lipid dynamics at a site in the groove between transmembrane domain 3,5 and 7 that has been suggested to regulate function by inhibition and which is also a hot-spot for disease mutations. This presumed inhibitory site showed state-dependent protein-lipid interactions; while POPE lipids and cholesterol embedded the sodium-bound state, the potassium-bound state had a higher presence of POPS and POPI lipids. Upon exploring disease mutant simulations at the protein-lipid interface, we found altered lipid interactions at the mutation sites, in particular we observed stable interactions with PS and PE lipids. These mutant-induced lipid interactions resulted in decreased protein dynamics, which possibly reflect resistance to normal transport behavior. Hence, our simulation approach constitutes a potential tool to better understand shifts in protein-lipid interactions during normal cycling of the Na,K ATPase transporter, and also to better understand the molecular basis of disease mutations.

Trophic Interactions, Management Trade-Offs and Climate Change: The Need for Adaptive Thresholds to Operationalize Ecosystem Indicators

Ecosystem-based management (EBM) is commonly applied to achieve sustainable use of marine resources. For EBM, regular ecosystem-wide assessments of changes in environmental or ecological status are essential components, as well as assessments of the effects of management measures. Assessments are typically carried out using indicators. A major challenge for the usage of indicators in EBM is trophic interactions as these may influence indicator responses. Trophic interactions can also shape trade-offs between management targets, because they modify and mediate the effects of pressures on ecosystems. Characterization of such interactions is in turn a challenge when testing the usability of indicators. Climate variability and climate change may also impact indicators directly, as well as indirectly through trophic interactions. Together, these effects may alter interpretation of indicators in assessments and evaluation of management measures. We developed indicator networks - statistical models of coupled indicators - to identify links representing trophic interactions between proposed food-web indicators, under multiple anthropogenic pressures and climate variables, using two basins in the Baltic Sea as a case study. We used the networks to simulate future indicator responses under different fishing, eutrophication and climate change scenarios. Responsiveness to fishing and eutrophication differed between indicators and across basins. Almost all indicators were highly dependent on climatic conditions, and differences in indicator trajectories >10% were found only in comparisons of future climates. In some cases, effects of nutrient load and climate scenarios counteracted each other, altering how management measures manifested in the indicators. Incorporating climate change, or other regionally non-manageable drivers, is thus necessary for an accurate interpretation of indicators and thereby of EBM measure effects. Quantification of linkages between indicators across trophic levels is similarly a prerequisite for tracking effects propagating through the food web, and, consequently, for indicator interpretation. Developing meaningful indicators under climate change calls for iterative indicator validations, accounting for natural processes such as trophic interactions and for trade-offs between management objectives, to enable learning as well as setting target levels or thresholds triggering actions in an adaptive manner. Such flexible strategies make a set of indicators operational over the long-term and facilitate success of EBM.

Interaction Rates of Group-III and Group-V Impurities with Intrinsic Point Defects in Irradiated Si and Ge

A comparative study of interactions of shallow impurities with primary defects in oxygen- and carbon-lean moderately doped Si and Ge subjected to irradiation with 0.9 MeV electrons, 60Co gamma-rays, and 15 MeV protons at room temperature is presented and discussed. For the quantitative characterization of such interactions, changes in the total concentration of the original shallow group-V donor or group-III acceptor impurities in the irradiated materials are determined by Hall effect measurements over a wide temperature range. Losses of the shallow donor or acceptor states in the irradiated Si and Ge are indicative of their removal rates that can be used for estimation of production rates of primary defects interacting with the dopants. Some important factors affecting the interactions between primary defects and shallow impurities in Si and Ge are highlighted.

Interaction between DNA and Drugs Having Protonable Basic Groups: Characterization through Affinity Constants, Drug Release Kinetics, and Conformational Changes

This paper reports the in vitro characterization of the interaction between the phosphate groups of DNA and the protonated species of drugs with basic groups through the determination of the affinity constants, the reversibility of the interaction, and the effect on the secondary structure of the macromolecule. Affinity constants of the counterionic condensation DNA–drug were in the order of 106. The negative electrokinetic potential of DNA decreased with the increase of the proportion of loading drugs. The drugs were slowly released from the DNA–drug complexes and had release kinetics consistent with the high degree of counterionic condensation. The circular dichroism profile of DNA was not modified by complexation with atenolol, lidocaine, or timolol, but was significantly altered by the more lipophilic drugs benzydamine and propranolol, revealing modifications in the secondary structure of the DNA. The in vitro characterization of such interactions provides a physicochemical basis that would contribute to identify the effects of this kind of drugs in cellular cultures, as well as side effects observed under their clinical use. Moreover, this methodology could also be projected to the fields of intracellular DNA transfection and the use of DNA as a carrier of active drugs.

Modeling Binding Affinity of Pathological Mutations for Computational Protein Design.

An important aspect of protein functionality is the formation of specific complexes with other proteins, which are involved in the majority of biological processes. The functional characterization of such interactions at molecular level is necessary, not only to understand biological and pathological phenomena but also to design improved, or even new interfaces, or to develop new therapeutic approaches. X-ray crystallography and NMR spectroscopy have increased the number of 3D protein complex structures deposited in the Protein Data Bank (PDB). However, one of the more challenging objectives in biological research is to functionally characterize protein interactions and thus identify residues that significantly contribute to the binding. Considering that the experimental characterization of protein interfaces remains expensive, time-consuming, and labor-intensive, computational approaches represent a significant breakthrough in proteomics, assisting or even replacing experimental efforts. Thanks to the technological advances in computing and data processing, these techniques now cover a vast range of protocols, from the estimation of the evolutionary conservation of amino acid positions in a protein, to the energetic contribution of each residue to the binding affinity. In this chapter, we review several existing computational protocols to model the phylogenetic, structural, and energetic properties of residues within protein-protein interfaces.

Graph-Based Approach for Spatial Heterogeneity Analysis in Tumor Microenvironment

Introduction/ Background The interaction between tumor and surrounding microenvironment (TME) is recognized as playing an important role in the progression of the disease. Understanding of the interaction between tumor and immune system is the focus of several studies dedicated to the improvement of cancer immunotherapy effectiveness [1]. On the other hand, it has been shown that invasion and metastasis of breast tumors is influenced by collagen organization at the tumor-stromal interface [2]. The characterization of such interactions relies on an efficient spatial distribution quantification of TME. Graph-based analysis tools are the best suitable to answer this question as they have the ability to represent spatial arrangements and neighborhood relationships of different tissue components [3]. Aims In this work, we propose a novel approach to characterize the spatial relationships between cancer cells and TME components in breast tumors, using graph theory and sparse sets’ mathematical morphology (MM). The tools of morphology on graphs were first used in [4] to study the neighborhood relationships between cells in germinal centers from lymph nodes, then in [5] for semantic spatial configuration modeling in histopathology. In our study, we propose new morphological descriptors characterizing the tumor architecture and the interactions with TME cells. Methods Towards a better evaluation and understanding, we use simulated data of different breast tumor types , , where locations of cancer nuclei (CN), fibroblasts (synthesizers of collagen, FN), and lymphocytes (LN) are already known. In order to set neighborhood relationships between different cells, Delaunay graph [3] is first reconstructed on all cells, and alpha-shape filter [5] is applied to circumvent border effects, giving new graph denoted G . The designed features are extracted basically from two different morphological operations. The first operation is composed of successive morphological erosions [4] applied to the subgraph induced by CN (denoted SGC, ), repeated until the subgraph is null. The curve given by the number of CN in terms of erosions provides 3 significant characteristics : I) The origin slope describes the number of CN on the boundary of tumor aggregates (TA) and, thus, the tumor-stromal interface ; II) The area under curve (AUC) reflects the density within TAs, and III) the number of iterations outlines the morphologic radius of the largest TA and, consequently, the geodesic distance of the farthest tumor cell from LN and/or FN. The second morphological operation is composed of successive morphological dilations applied to SGC with non-overlapping control of labeled connected-components . The goal behind this operation is to investigate the TME cells surrounding each TA. The ratio between the number of LN and the number of CN, and the means of the Euclidean and the geodesic distances of LN from CN on the boundary are calculated for each TA . Results In this work, we have briefly presented a conceptual framework for analyzing the architecture of breast tumors and the interactions with the surrounding microenvironment. New graph-based features were proposed to characterize the spatial distribution of TME components and were tested on simulated data. In our future works, we will include adipose tissue [6], blood vessels and endothelial cells. We will also focus on the anisotropic characterization of collagen, and test the approach on real dataset.

Intracellular expression of camelid single-domain antibodies specific for influenza virus nucleoprotein uncovers distinct features of its nuclear localization.

Perturbation of protein-protein interactions relies mostly on genetic approaches or on chemical inhibition. Small RNA viruses, such as influenza A virus, do not easily lend themselves to the former approach, while chemical inhibition requires that the target protein be druggable. A lack of tools thus constrains the functional analysis of influenza virus-encoded proteins. We generated a panel of camelid-derived single-domain antibody fragments (VHHs) against influenza virus nucleoprotein (NP), a viral protein essential for nuclear trafficking and packaging of the influenza virus genome. We show that these VHHs can target NP in living cells and perturb NP's function during infection. Cytosolic expression of NP-specific VHHs (αNP-VHHs) disrupts virus replication at an early stage of the life cycle. Based on their specificity, these VHHs fall into two distinct groups. Both prevent nuclear import of the viral ribonucleoprotein (vRNP) complex without disrupting nuclear import of NP alone. Different stages of the virus life cycle thus rely on distinct nuclear localization motifs of NP. Their molecular characterization may afford new means of intervention in the virus life cycle. Many proteins encoded by RNA viruses are refractory to manipulation due to their essential role in replication. Thus, studying their function and determining how to disrupt said function through pharmaceutical intervention are difficult. We present a novel method based on single-domain-antibody technology that permits specific targeting and disruption of an essential influenza virus protein in the absence of genetic manipulation of influenza virus itself. Characterization of such interactions may help identify new targets for pharmaceutical intervention. This approach can be extended to study proteins encoded by other viral pathogens.

Quantifying Protein Adsorption and Function at Nanostructured Materials: Enzymatic Activity of Glucose Oxidase at GLAD Structured Electrodes

Nanostructured materials strongly modulate the behavior of adsorbed proteins; however, the characterization of such interactions is challenging. Here we present a novel method combining protein adsorption studies at nanostructured quartz crystal microbalance sensor surfaces (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical methods (cyclic voltammetry CV) allowing quantification of both bound protein amount and activity. The redox enzyme glucose oxidase is studied as a model system to explore alterations in protein functional behavior caused by adsorption onto flat and nanostructured surfaces. This enzyme and such materials interactions are relevant for biosensor applications. Novel nanostructured gold electrode surfaces with controlled curvature were fabricated using colloidal lithography and glancing angle deposition (GLAD). The adsorption of enzyme to nanostructured interfaces was found to be significantly larger compared to flat interfaces even after normalization for the increased surface area, and no substantial desorption was observed within 24 h. A decreased enzymatic activity was observed over the same period of time, which indicates a slow conformational change of the adsorbed enzyme induced by the materials interface. Additionally, we make use of inherent localized surface plasmon resonances in these nanostructured materials to directly quantify the protein binding. We hereby demonstrate a QCM-D-based methodology to quantify protein binding at complex nanostructured materials. Our approach allows label free quantification of protein binding at nanostructured interfaces.

Nanodisc-based Co-immunoprecipitation for Mass Spectrometric Identification of Membrane-interacting Proteins

Proteomic identification of protein interactions with membrane associated molecules in their native membrane environment pose a challenge because of technical problems of membrane handling. We investigate the possibility of employing membrane nanodiscs for harboring the membrane associated molecule to tackle the challenges. Nanodiscs are stable, homogenous pieces of membrane with a discoidal shape. They are stabilized by an encircling amphipatic protein with an engineered epitope tag. In the present study we employ the epitope tag of the nanodiscs for detection and co-immunoprecipitation of interaction partners of the glycolipid ganglioside GM1 harbored by nanodiscs. Highly specific binding activity for nanodisc-GM1 immobilized on sensorchips was observed by surface plasmon resonance in culture media from enterotoxigenic Escherischia coli. To isolate the interaction partner(s) from enterotoxigenic Escherischia coli, GM1-nanodiscs were employed for co-immunoprecipitation. The B subunit of heat labile enterotoxin was identified as a specific interaction partner by mass spectrometry, thus demonstrating that nanodisc technology is useful for highly specific detection and identification of interaction partners to specific lipids embedded in a membrane bilayer.

Excimer-Based Peptide Beacons: A Convenient Experimental Approach for Monitoring Polypeptide−Protein and Polypeptide−Oligonucleotide Interactions

While protein-polypeptide and nucleic acid-polypeptide interactions are of significant experimental interest, quantitative methods for the characterization of such interactions are often cumbersome. Here we described a relatively simple means of optically monitoring such interactions using excimer-based peptide beacons (PBs). The design of PBs is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed with macromolecular targets. Using this binding-induced folding to segregate two pyrene moieties and therefore inhibit excimer formation, we have produced PBs directed against both anti-HIV antibodies and the retroviral transactive response (TAR) RNA hairpin. For both polypeptides, target recognition is accompanied by a roughly 2-fold decrease in excimer emission, thus allowing the detection of their respective targets at concentrations of a few nanomolar. Because excimer emission requires the formation of a tight, precisely oriented pyrene dimer, even relatively trivial binding-induced segregation reduces fluorescence significantly. This suggests that the PB approach will be suitable for monitoring a wide range of peptide-macromolecule recognition events. Moreover, the synthesis of excimer-based PBs utilizes commercially available modified pyrenes in a simple and well-established protocol, making the approach well suited for routine laboratory applications.

Overview of the Quantitation of Protein Interactions

The biological function of many proteins involves reversible interactions with other proteins, nucleic acids, or other non-protein ligands. Such interactions play many different roles in a wide range of cellular processes. A few examples are: (1) storing or transporting key metabolites (e.g., O(2) storage by myoglobin); (2) forming and maintaining the quaternary structure of multi-subunit enzymes; (3) specific binding and recognition events (antigen-antibody, hormone-receptor, transcription factor-promoter); and (4) self-assembly of large structures (microtubules, chromatin). Thus, the quantitative characterization of such interactions represents an important part of understanding the function of such proteins and their role in these cellular events. This unit sets the tone for the rest of the chapter, and gives important information necessary to understand some of the topics that will be covered in future supplements, such as sedimentation equilibrium (analytical and micro-preparative), surface plasmon resonance (SPR), size-exclusion chromatography (SEC) with on-line light scattering, and chemical cross-linking.

Characterization of Si(100) sputtered with low energy argon

The near surface structure of low energy (0.5-1.5 keV) argon bombarded Si(100) was characterized using medium energy ion scattering and high resolution X-ray photoemission spectroscopy. The ion induced lattice damage, distribution and redistribution of incorporated argon and silicon carbide formed during the dynamic mixing process are directly and non-destructively measured and depth-profiled in the sub-nanometre scale. The results capture many details of low energy ion interaction with Si in the near surface and address the capability for direct and non-destructive characterization of such interactions in the sub-nanometre scale.

A photochemically induced dynamic nuclear polarization study of denatured state of lysozyme

Photochemically induced dynamic nuclear polarization (photo-CIDNP) techniques have been used to examine denatured states of lysozyme produced under a variety of conditions. 1H CIDNP difference spectra of lysozyme denatured thermally, by the addition of 10 M urea, or by the complete reduction of its four disulfide bonds were found to differ substantially not only from the spectrum of the native protein but also from that expected for a completely unstructured polypeptide chain. Specifically, denatured lysozyme showed a much reduced enhancement of tryptophan relative to tyrosine than did a mixture of blocked amino acids with the same composition as the intact protein. By contrast, the CIDNP spectrum of lysozyme denatured in dimethyl sulfoxide solution was found to be similar to that expected for a random coil. It is proposed that nonrandom hydrophobic interactions are present within the denatured states of lysozyme in aqueous solution and that these reduce the reactivity of tryptophan residues relative to tyrosine residues. Characterization of such interactions is likely to be of considerable significance for an understanding of the process of protein folding.

Interfacial Characterization of Epoxy-bonded Maraging Steel

Abstract Adhesion phenomena involving polymer-metal interfaces have become a central subject for research and technological development, especially in view of adhesive bonding of metallic components in primary load-bearing structures. Since adhesion implies that some sort of chemical and/or physical interactions take place between the metal adherend and the polymeric adhesive, a great deal of attention has been devoted to identification and characterization of such interactions and to the development of techniques to study them. Among the most widely used methods for interfacial studies are: ESCA (Electron Spectroscopy for Chemical Analysis), AES (Auger Electron Spectroscopy), SIMS (Secondary Ion Mass Spectroscopy), IR spectroscopy, optical microscopy, and electron microscopy.