Binary Protein Research Articles

X-ray Structure of the Human Karyopherin RanBP5, an Essential Factor for Influenza Polymerase Nuclear Trafficking

Here, we describe the crystal structures of two distinct isoforms of ligand-free human karyopherin RanBP5 and investigate its global propensity to interact with influenza A virus polymerase. Our results confirm the general architecture and mechanism of the IMB3 karyopherin-β subfamily whilst also highlighting differences with the yeast orthologue Kap121p. Moreover, our results provide insight into the structural flexibility of β-importins in the unbound state. Based on docking of a nuclear localisation sequence, point mutations were designed, which suppress influenza PA-PB1 subcomplex binding to RanBP5 in a binary protein complementation assay.

A systems toxicology approach to compare the heavy metal mixtures (Pb, As, MeHg) impact in neurodegenerative diseases

Conventional toxicological risk assessment methods mainly working on single chemicals that fail to adequately address the simultaneous exposure and their potential toxicity in humans. We herein investigated the toxic heavy metals lead (Pb), arsenic (As), and methylmercury (MeHg) and their binary mixtures role in neurodegenerative diseases. To characterize the toxicity of metal mixtures at the molecular level, we established a non-animal omics-based organ relevant cell model system. The obtained experimental data was refined by using the statistical and downstream functional analysis. The protein expression information substantiates the previous findings of single metal (Pb, As, and MeHg) induced alterations to mitochondrial dysfunction, oxidative stress, mRNA splicing, and ubiquitin system dysfunction relation to neurodegenerative diseases. The functional downstream analysis of single and binary mixtures protein data is presented in a comparative manner. The heavy metals mixtures’ outcome showed significant differences in the protein expression compared to single metals that indicate metal mixtures exposure is more hazardous than single metal exposure. These results suggest that more comprehensive strategies are needed to improve the mixtures risk assessment in the future.

Mapping Interactions among Cell-Free Expressed Zika Virus Proteins.

The rapid spread of arthropod-borne Zika virus poses a serious public health threat that calls for effective ways of controlling and treating viral infection. This in turn necessitates better understanding of the mechanisms of virus assembly and its interaction with the host cells. In order to facilitate such efforts, we developed a new multihost expression vector pmCellFree that allows rapid and multiplexed production of ZIKV proteins in any in vitro translation system as well as in mammalian cells. Using a combination of in vitro expression in Leishmania cell-free system and AlphaLISA interaction assay, pairwise protein interactions of all ZIKV proteins were systematically tested. We identified thirty-three intraviral binary protein interactions, of which 13 interactions are novel. These findings were further validated by expressing selected protein pairs in mammalian HEK293T cell line and assessing their interactions in the cellular lysate. The results of these interaction assays were identical to those obtained with in vitro expressed proteins. The observed novel protein-protein interactions were further validated using a pulldown assay. The unrevealed novel protein interactions may point to the previously unappreciated complexity of the ZIKV assembly process and may play an important role in the infection process. These interactions may represent new targets for antiviral drug development.

Structural Insight into Binary Protein Metal-Organic Frameworks with Ferritin Nanocages as Linkers and Nickel Clusters as Nodes.

Metal-organic frameworks (MOFs) hold great promise for numerous applications. However, proteins, carriers of biological functions in living systems, have not yet been fully explored as building blocks for the construction of MOFs. This work presents a strategy for the fabrication of binary MOFs. Considering octahedral ferritin symmetry, four His2 (His-His) motifs were first incorporated into the exterior surface of a ferritin nanocage near each C4 channel, yielding protein linkers with multiple metal-binding sites (bisH-SF). Secondly, by adding nickel ions to bisH-SF solutions triggers the self-assembly of ferritin nanocages into a porous 3D crystalline MOF with designed protein lattice, where two adjacent ferritin molecules along the C4 symmetry axes are bridged by four dinuclear or tetranuclear nickel clusters depending on Ni2+ concentration. This work provides a simple approach for precise control over a binary protein-metal crystalline framework, and the resulting MOFs exhibited inherent ferroxidase activity and peroxidase-like catalytic activity.

Label-free surface-enhanced Raman spectroscopy detection of absorption manner of lysozyme based on nanodots arrays

Lysozyme plays an important role in the innate immune system of animal or human body, thus, it is vital to detect it using a high sensitivity and efficiency approach in clinic. Nanostructure-based Surface-enhanced Raman scattering (SERS) substrate is often employed to detect biomolecules. In this study, a nanodots array structure, which was utilized as a micro-nano hybrid SERS substrate, was fabricated using second-scan scratching strategy with AFM-probe. The dependence of the nanodots structure on the machining parameters such as applied normal loads and scratching angles were investigated. The effects of the nanostructure feature sizes and the Au film thickness on the measured SERS spectra that tested using Rhodamine 6G (R6G) were studied. In addition, different drying temperatures and concentrations of NaNO3 solution were used to study the absorption manner of lysozyme on an Ag substrate. The SERS spectra of a binary protein mixture, which includes lysozyme and insulin, were also investigated based on the fabricated nanostructures. Results showed that the substrates fabricated using AFM nanomachining approach may promote the SERS application in biologic field.

A Self‐Assembled Binary Protein Model Explains High‐Performance Salivary Lubrication from Macro to Nanoscale

AbstractSalivary pellicle, a spontaneously formed, intricate architecture in the human oral cavity, is a high‐performance bio‐lubricant that coats and protects biological surfaces with varying elastic modulus against frictional damage. Although salivary lubrication underpins the fundamentals of human feeding and speech, the peculiar molecular mechanism behind such lubrication properties remains elusive. For the first time, this work demonstrates a binary model comprised of salivary proteins, mucin, and lactoferrin (LF), forming an electrostatically driven, multilayered self‐assembly that exhibits a lubrication behavior closely resembling that of human saliva, from macro to nanoscale. The multiscale tribological analysis with applied forces ranging from 1 N to 1 nN, supported by real‐time self‐assembly monitoring on hydrophilic and hydrophobic substrates differentially resolves the distinct roles played by the salivary proteins of this proposed lubricating model. Evidences reveal that hydrated mucin controls the macromolecular viscous lubrication entrapping water molecules in the mucinous network and LF acts as a “molecular glue” between mucin–mucin and mucin–surface, latter aiding boundary lubrication. This study puts forward an unprecedented molecular model that explains the synergistic lubrication by salivary components. These results can aid into the design routes for synthesizing highly efficacious nature‐inspired aqueous lubricants for future biomedical applications and nutritional technologies.

Protein interactome network analysis predicts novel breast cancer candidate genes with molecular signatures

Abstract Background Advancement in molecular biology research has allowed the measurement of multiomics data points from a single tumor biopsy sample in a reasonable time frame for making significant clinical decisions. There have been tremendous advances from the bioinformatics and systems biology perspective to analyse integrated multi‐scale analysis of the data will prove invaluable for a combined systems‐level understanding of the important biological network processes contributing to the initiation, progression and management of cancer. Cancer networks have greater challenge due to the aberrant functions of hundreds of genes that are translated to altered protein function within each cancer cell, to understand this complexity. Network-based methods have been used to analyse the complexity molecular interactions in the cell and can contribute to prediction of candidate genes responsible for cancer. Methods HI-II- 14 is the largest experimentally determined binary protein – protein interaction map is used for the study. These interactions were mapped to NCBI Entrez gene ID and excluded self-loop and redundant interactions. The known human breast cancer genes were obtained from the Sanger Cancer Gene Census which is a comprehensive catalogue of genes implicated in breast cancer and from Uniprot database. In total, we obtained 913 genes from both for analyses. The genes degree of connections and the centrality in the protein -protein networks were considered. The genome-wide mutation datasets of breast cancer were downloaded from TCGA as maf files. For each gene, we obtained the mutation frequency for each gene is calculated which is defined as the number of mutated samples divided by the total number of samples. The frequency of the top 100 ranked genes and the bottom 100 ranked genes were compared by Wilcox rank sum test. Results In this study, HI-II-14 human protein-protein interactome network is applied to prediction of novel breast cancer genes. Conclusions Our study suggests integrating interactome networks with multiomics datasets could provide novel insights into breast cancer-associated genes and underlying molecular mechanisms. Legal entity responsible for the study M. Peer Mohammed. Funding Has not received any funding. Disclosure The author has declared no conflicts of interest.

Sequential binary protein patterning on surface domains of thermo-responsive polymer blends cast by horizontal-dipping

We characterize an approach enabling dual protein positioning over broad polymer areas based on subsequent selective adsorption of two fluorescently labelled lectins, Concanavalin A (Con A) and Lentil Lectin (LcH), on self-assembled gradient patterns of thermoresponsive poly(N-isopropyl acrylamide) (PNIPAM) and polystyrene (PS) polymers blend, prepared by horizontal dipping technique. The film morphologies of gradient samples prior dual selective protein adsorption are mapped with scanning microscopy (AFM) and secondary ion mass spectrometry (ToF-SIMS), whereas adsorbed proteins are imaged with fluorescence microscope. ToF-SIMS analysis reveals surface composition consisting of PNIPAM-rich domains in PS-rich matrix. The two-step protein adsorption experiment results in selective adsorption of Con A and LcH to PNIPAM- and PS-rich phases, respectively. Integral geometry approach is used to compare quantitatively morphology of polymer patterns varied in domain size due to horizontal dipping casting. Minkowski measures are also used to compare quantitatively fluorescence micrographs of protein patches with SIMS images of original isotropic polymer patterns. It confirms that PNIPAM domains size increases with increasing speed. Further, Minkowski analysis unveiled that adsorbed proteins cover about 60–70% of polymer surface. What is more fluorescence micrographs acknowledge both no lectins contamination and no adsorption to interphase areas. Additionally, protein displacement effect is observed.

Multi-Scale Simulations Yield Insight into Protein Diffusion and Stability in Crowded Environments

In living cells, proteins operate in a highly crowded environment where a large fraction of the volume is filled by macromolecules. Despite intensive research, the effects of macromolecular crowding on protein mobility, stability, and ultimately function have not yet been fully elucidated. The need for a detailed microscopic understanding calls for insights from computer simulations; however, the wide spread of time- and length scales involved in crowding poses a serious challenge for current in silico approaches. Our novel multi-scale scheme, combining lattice Boltzmann molecular dynamics (LBMD) with all-atom replica exchange simulations, bridges a gap between existing simulation techniques and provides a computational framework for exploring protein diffusion and stability in the intracellular environment. Our LBMD simulations, employing the OPEP coarse-grain model, reproduce the experimentally observed diffusion slowdown in several crowded binary protein mixtures and allow us to characterize the timescales of protein reshuffling as well as the typical geometries of local packing around the proteins. Furthermore, we use our computational scheme to shed light on the unfolding of superoxide dismutase 1 (SOD1) in crowded conditions, a process implicated in amyotrophic lateral sclerosis (ALS). We show that the thermal stability of a SOD1 barrel depends on its interactions with the surrounding proteins. As a consequence, the different states of local packing result in crowding-induced heterogeneity of SOD1 thermal stabilities. Thus, our results highlight the importance of considering the local environment when investigating protein function in the cytoplasm.

Illuminating SUN2/KASH Hetero-Complex Formation Within the Nuclear Envelope of Living Cells with Dual-Color Time-Shifted MSQ

The nuclear envelope (NE) is a cellular compartment consisting of an inner and outer nuclear membrane separated by the perinuclear space. Research on cellular mechanotransduction identified the nucleus as a mechanosensor, which responds to the mechanical properties of the extracellular environment. Force transmission across the NE into the nucleoplasm is facilitated by linker of nucleoskeleton-and-cytoskeleton (LINC) complexes, which are conserved molecular bridges formed by the transluminal physical interaction of the outer and inner nuclear membrane KASH and the SUN proteins, respectively. Mammals encode six KASH proteins and five SUN proteins. In vitro, KASH proteins bind to the domain interfaces of trimeric SUN2 proteins resulting in the formation of a SUN2/KASH hetero-hexamer. Recently, we extended the application of single-color fluorescence fluctuation spectroscopy (FFS) to quantify protein oligomerization within the NE of living cells by developing mean segmented Q-factor (MSQ) and time-shifted MSQ (tsMSQ) to address experimental challenges unique to the environment of the NE. Using FFS, we demonstrated that SUN2 trimerizes in vivo, whereas the luminal SUN-binding KASH peptide of the KASH protein nesprin-2 (KASH2) remains monomeric. To be able to identify the oligomerization of SUN2/KASH within the NE of living cells, we describe here the development of dual-color (DC) tsMSQ. We first validated DC tsMSQ using nuclear envelope proteins tagged with EGFP and mCherry as model systems. Finally, we applied DC tsMSQ to detect the formation of hetero-complexes of EGFP-tagged SUN2 and mCherry-tagged KASH2. The development of DC tsMSQ will enable future efforts aimed at the mechanistic dissection of LINC complex assembly and its regulation within the NE as well as investigations of binary protein systems within this important sub-cellular compartment. This work has been supported by a grant from the NIH (R01 GM64589).

Peptidomimetics Targeting Protein-Protein Interactions for Therapeutic Development.

Interactions between proteins play a key role in nearly all cellular process, and therefore, its dysregulation may lead to many different types of cellular dysfunctions. Hence, pathologic Protein-Protein Interactions (PPIs) constitute highly attractive drug targets and hold great potential for developing novel therapeutic agents for the treatment of incurable human diseases. Unfortunately, the identification of PPI inhibitors is an extremely challenging task, since traditionally used small molecules ligands are mostly unable to cover and anchor on the extensive and flat surfaces that define those binary protein complexes. In contrast, large biomolecules such as proteins or peptides are ideal fits for this so-called "undruggable" sites. However, their poor pharmacokinetic properties have also limited their applications as therapeutics. In this context, peptidomimetic molecules have emerged as an alternative and viable solution to this problem, since they conserve the architectural and structural features of peptides and also exhibit substantially improved pharmacokinetic profiles. In the last decades, a wide array of chemical approaches granting access to conformationally constrained peptides with substantially improved pharmacokinetic profiles have been described, with a special focus on those affording stapled peptides and allowing large-scale macrocyclizations. These peptidomimetic molecules have been successfully applied to target a plethora of biological hosts, which highlights their promising future as novel therapeutics for the treatment of incurable human diseases.

Metal-Assisted Assembly of Protein Containers Loaded with Inorganic Nanoparticles.

Protein containers are suitable building blocks for bioinorganic materials. Here, we show that high concentrations of magnesium ions induce the formation of a unitary protein scaffold, whereas low magnesium concentration leads to a binary protein scaffold. The molecular interactions in the protein scaffold were characterized with X-ray crystallography to high resolution. We show that the unitary framework can be applied for the assembly of inorganic nanoparticles such as metal oxides into highly ordered bioinorganic structures. Our work emphasizes the structural tunability of protein-container-based materials, important for adjusting emerging properties of such materials.

Gas‐assisted low‐field magnetic separation for large scale continuous magnetic bio‐separation process

In this study, the gas‐assisted low‐field magnetic separation (GAMS) was proposed for continuous large‐scale magnetic bio‐separation process. Magnetic recovery processes in selective separation of binary mixed proteins by magnetic microspheres were selected as a model. Basing on magnetic recovery efficiency of the batch GAMS for this model, the continuous GAMS with an amplifying small pilot scale was further researched. The continuous throughput of GAMS for adsorption, washing, and desorption step reached 12 L/h, 12 L/h, and 18 L/h with an adsorbent recovery above 95%, respectively. Furthermore, magnetic particles via the top magnetic roller could be well‐separated from free proteins with good floatability via the bottom outlet of flotation column, and bubbling operation did not cause the significant denaturation of target protein. Compared with classic high gradient magnetic separation, GAMS had the advantages of easy scale‐up, easy continuous operation, and low‐cost, showing great potential for large‐scale magnetic bio‐separation process. © 2018 American Institute of Chemical Engineers AIChE J, 65: 175–183, 2019

Mosquito-larvicidal binary toxin receptor protein (Cqm1): crystallization and X-ray crystallographic analysis.

Cqm1 from Culex quinquefasciatus has been identified as the receptor for Lysinibacillus sphaericus binary toxin (BinAB). It is an amylomaltase that is presented on the epithelial membrane in the larval midgut through a glycosyl-phosphatidylinositol anchor. The active core of this protein (residues 23-560) was overexpressed in Escherichia coli, purified and successfully crystallized by the sitting-drop vapor-diffusion method using D-arabinose and CaCl2 as additives, as identified using high-throughput differential scanning fluorimetry analysis. X-ray diffraction data were collected to a resolution of 2.8 Å using a laboratory X-ray source. The crystals had the symmetry of space group P212121, with unit-cell parameters a = 191.3, b = 205.3, c = 59.0 Å and with four monomers in the asymmetric unit. Structure refinement is in progress. This is the first structure report for a binary toxin receptor and for a member of the GH13_17 subfamily in the CAZy database.

Efficient adsorptive extraction materials by surface protein‐imprinted polymer over silica gel for selective recognition/separation of human serum albumin from urine

ABSTRACTIn this study, we report the development of adsorptive extraction materials by surface protein‐imprinted polymers (MIPs) over silica gel for selective recognition/separation of human serum albumin (HSA) from urine. The HSA‐imprinted polymers prepared on silica particle had at interface between the silica gel and different MIPs greatly produced enrichment for the binding of protein from the urine. The solid‐phase extraction of the optimized polymer layer was prepared by copolymerization of methacrylic acid (MAA), acrylamide (AAm), and dimethylaminoethylmethacrylate (DMAEMA) and a crosslinker methylenebisacrylamide (MBA) at the mole ratio of 1:158:88 (T:M:C) and showed moderate affinity (<104 order M−1) toward target protein HSA and selectivity. Four analogues, egg white albumin (EWA), bovine serum albumin (BSA), lysozyme (Lyz), and creatinine (Cre) were selected to study the binding efficiency of MIPs in single and binary protein solutions. We studied the influence on recognition ability for HSA and found that prepolymer mixture and matrix flexibility of the optimized thin polymer layer (35 ± 10 nm) on the submicrosilica particles. The high‐binding affinity (QMIP, 86.7 mg g−1) and fast kinetics (180 min) were observed for this synthesized HSA‐MIP when compared with other reported HSA‐MIPs in surface imprinting (5.9 and 11.3 mg g−1) and epitope surface imprinting (46.6 mg g−1) methods. We demonstrated the application in real and synthetic urine samples that the approach allowed the efficient adsorption of HSA in real urine (129.48 mg g−1) is almost double to the binding of HSA in synthetic urine (67.84 mg g−1). Apart from this, only minor interference of Cre (2.74 mg g−1) was observed, eventhough Cre is the final metabolite in urine. These adsorptive submicrosilica materials have potential in the pharmaceutical industry and clinical analysis applications. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 46894.

Gateway-Compatible Yeast One-Hybrid and Two-Hybrid Assays.

In the first section of this introduction, we provide background information for yeast two-hybrid (Y2H) assays that provide a genetic method for the identification and analysis of binary protein-protein interactions and that are complementary to biochemical methods such as immunoprecipitation. In the second section, we discuss yeast one-hybrid (Y1H) assays that provide a "gene-centered" (DNA-to-protein) genetic method to identify and study protein-DNA interactions between cis-regulatory elements and transcription factors (TFs). This method is complementary to "TF-centered" (protein-to-DNA) biochemical methods such as chromatin immunoprecipitation.

A toxin-antitoxin system is essential for the stability of mosquitocidal plasmid pBsph of Lysinibacillus sphaericus

Lysinibacillus sphaericus C3-41 carries a large low-copy-number plasmid pBsph, which encodes binary toxin proteins. Our previous study found that the transcriptional activator TubX plays an important role in the newly identified type Ⅲ TubRZC replication/partition system in pBsph, and that a vector consisting of tubRZC and tubX is not as stable as pBsph, indicating the presence of other maintenance module(s). In this study, we identified that orf9 and orf10 are necessary for the stability of pBsph by a series of deletion and complementation experiments. Bioinformatics analysis showed that ORF9 contains a PIN domain of VapBC toxin-antitoxin (TA) system, whereas ORF10 share no significant sequence similarity to any of the characterized antitoxins in the database. Further studies revealed that orf9 and orf10 are transcribed as an operon. The overexpression of ORF9 repressed the growth of both Escherichia coli and L. sphaericus, which can be alleviated by overexpression of ORF10. The deletion of orf10 individually or orf9-10 together resulted a decrease on plasmid stability which was restored by the complementation of corresponding gene(s), suggesting that ORF10 plays an important role in plasmid stability. In addition, it was found the plasmid stability is related with the transcription level of tubRZ, and overexpression of TubRZ could neutralize the negative effect on plasmid stability caused by the deletion of orf9-orf10. Moreover, the recombinant vector containing tubRZC, tubX and orf9-10 was more stable than the ones containing only tubRZC and either tubX or orf9-10. The data indicate that the plasmid maintenance system on pBsph includes orf9-orf10 TA system.

Conformational and Organizational Insights into Serum Proteins during Competitive Adsorption on Self-Assembled Monolayers.

Physicochemical interactions of proteins with surfaces mediate the interactions between the implant and the biological system. Surface chemistry of the implant is crucial as it regulates the events at the interface. The objective of this study was to explore the performance of modified surfaces for such interactions relevant to various biomedical applications. Because of a wide range of surface wettability, we aimed to study protein behavior (i.e., conformational changes and their packing) during competitive protein adsorption. Three serum proteins (bovine serum albumin, BSA; fibrinogen, FB; and immunoglobulin G, IgG) were tested for their conformational changes and orientation upon adsorption on hydrophilic (COOH and amine), moderately hydrophobic (mixed and hybrid), and hydrophobic (octyl) surfaces generated via silanization. Modified surfaces were characterized using Fourier-transform infrared spectroscopy, contact angle, and atomic force microscopy (AFM) techniques. Adsorbed masses of proteins from single and binary protein solutions on different surfaces were quantified along with their secondary structure analyses. Maximum adsorbed protein masses were found to be on negatively charged and hydrophobic (octyl) surfaces because of ionic and hydrophobic interactions between protein molecules and surfaces, respectively. Side-on and end-on orientations of adsorbed protein molecules were analyzed using theoretical and AFM analyses. We observed compact and elongated forms of BSA molecules on hydrophilic and hydrophobic surfaces, respectively. We further found a linear increase in the α-helix content of BSA and β-sheet contents of FB and IgG proteins with the increasing side-on (%)-oriented protein molecules on the surfaces. This indicates that side-on orientations of adsorbed FB and IgG lead to the formation of β-sheets. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was employed to quantify the protein types and their ratio in competitively adsorbed proteins on different surfaces. A theoretical analysis was also used to determine the % secondary structures of competitively adsorbed proteins from BSA/FB and BSA/IgG solutions, which very well agreed with experimental results. The competitive protein adsorption from both BSA/FB and BSA/IgG solutions was found to be entropy-driven, as revealed by thermodynamic studies performed using isothermal titration calorimetry.

Human alpha-defensin-1 protects cells from intoxication with Clostridium perfringens iota toxin.

Iota toxin is produced by Clostridium perfringens type E strains and associated with diarrhea in cattle and lambs. This binary protein toxin comprises the enzyme component iota a (Ia), which ADP-ribosylates G-actin, and the separate transport component iota b (Ib), which delivers Ia into the cytosol of target cells. Ib binds to cell receptors and forms biologically active toxin complexes with Ia, which cause rounding of adherent cells due to the destruction of the actin cytoskeleton. Here, we report that the human peptide α-defensin-1 protects cultured cells including human colon cells from intoxication with iota toxin. In contrast, the related ß-defensin-1 had no effect, indicating a specific mode of action. The α-defensin-1 did not inhibit ADP-ribosylation of actin by Ia in vitro. Pretreatment of Ib with α-defensin-1 prior to addition of Ia prevented intoxication. Additionally, α-defensin-1 protected cells from cytotoxic effects mediated by Ib in the absence of Ia, implicating that α-defensin-1 interacts with Ib to prevent the formation of biologically active iota toxin on cells. In conclusion, the findings contribute to a better understanding of the functions of α-defensin-1 and suggest that this human peptide might be an attractive starting point to develop novel pharmacological options to treat/prevent diseases associated with iota toxin-producing Clostridium perfringens strains.

The Yeast Three-Hybrid System for Protein Interactions.

Proteins rarely act alone as their functions tend to be regulated in vivo. Therefore, protein-protein interaction analyses provide key clues for understanding the complex biological processes in the living cell. Several techniques have been developed to elucidate the conformation of large protein complexes, dynamic protein complex rearrangement and transient protein interactions. Yeast two-hybrid system is a well-established method to analyze binary protein interactions. Here we describe a basic yeast three-hybrid method, which represents an additional refinement of the classical yeast two-hybrid system for analyzing further complex interactions among three proteins.