Nonspecific Protein Interactions Research Articles

Cluster Size and Quinary Structure Determine the Rheological Effects of Antibody Self-Association at High Concentrations.

The question of how nonspecific reversible intermolecular protein interactions affect solution rheology at high concentrations is fundamentally rooted in the translation of nanometer-scale interactions into macroscopic properties. Well-defined solutions of purified monoclonal antibodies (mAbs) provide a useful system with which to investigate the manifold intricacies of weak protein interactions at high concentrations. Recently, characterization of self-associating IgG1 antibody (mAb2) solutions has established the direct role of protein clusters on concentrated mAb rheology. Expanding on our earlier work with three additional mAbs (mAb1, mAb3, and mAb4), the observed concentration-dependent static light scattering and rheological data present a substantially more complex relationship between protein interactions and solution viscosity at high concentrations. The four mAb systems exhibited divergent correlations between cluster formation (size) and concentrated solution viscosities dependent on mAb primary sequence and solution conditions. To address this challenge, well-established features of colloidal cluster phenomena could be applied as a framework for interpreting our observations. The initial stages of mAb cluster formation were investigated with small-angle X-ray scattering (SAXS) and ensemble-optimized fit methods, to uncover shifts in the dimer structure populations which are produced by changes in mAb interaction modes and association valence under the different solution conditions. Analysis of mAb average cluster number and effective hydrodynamic radii at high concentrations revealed cluster architectures can have a wide range of fractal dimensions. Collectively, the static light scattering, SAXS, and rheological characterization demonstrate that nonspecific and anisotropic attractive intermolecular interactions produce antibody clusters with different quinary structures to regulate the rheological properties of concentrated mAb solutions.

A role for macromolecular crowding in off-target binding of therapeutic antibodies.

The nonspecific binding of certain therapeutic antibodies to tissues or to soluble biomolecules can accelerate their clearance from the circulation and undermine their benefit to patients. This article proposes that tandem amino acid repeat sequences in antibody hypervariable segments, particularly the complementarity determining regions (CDRs), can enhance this off-target binding. This hypothesis is based on two sets of observations. First, in a limited number of cases, antibodies with clusters of amino acid repeats in their CDRs have significantly higher clearance rates in experimental animals than otherwise identical antibodies without the repeats. Second, tandem amino acid repeats are abundant in intracellular hub proteins where they appear to promote the promiscuous binding of these proteins to a wide variety of other molecules. These nonspecific hub protein interactions are highly favored by the intense macromolecular crowding that permeates the cytoplasm. A survey of the variable region sequences of 137 antibodies in various stages of development revealed that 26 have at least one CDR containing a cluster of three closely spaced amino acid repeats. If the overall hypothesis is valid, then it suggests strategies for site-directed mutagenesis to improve pharmacokinetic behavior and for the design of more reliable in vitro binding assays to predict off-target binding in vivo.

Glycopolymer brushes with specific protein recognition property

ABSTRACTHere, we report the synthesis of glycopolymer brushes on the silicon substrate by interface‐mediated reversible addition‐fragmentation chain transfer polymerization of pentafluorophenyl acrylate as well as subsequent attachment of d‐glucosamine to determine specific and nonspecific protein interactions. The root‐mean‐square roughness of the glycopolymer brushes is only about 3.1 nm, indicating unstable distribution of the collapsed glycopolymer chains on the surface. The water contact angle of glycopolymer brush was determined as ∼53° indicating a hydrophilic behavior. The results of fluorescence microscopy studies indicated that the glycopolymer brushes have specific interaction with fluorescence‐labeled Concanavalin A and nonspecific interaction with fluorescence‐labeled bovine serum albumin. The glycopolymer brushes tested here are hoped to enlarge to our knowledge of recognition phenomena at the surface of polymer brushes. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 45238.

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide.

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

Single-Molecule Imaging of Transcription, Chromosome Organization, and DNA Repair in Live Bacteria

Protein-DNA interactions are critical to many important biological functions, from transcription and chromosome organization, to DNA replication and repair. To fully understand these processes we need to look at molecular details, such as the stoichiometries and binding kinetics of these proteins. However, focusing on the molecular level can miss the bigger picture; we also need to understand how protein-DNA interactions shape the global organisation of chromosomes and cause phenotypical changes over the whole cell.Using photoactivated single-molecule tracking in live bacteria allows proteins specifically bound to DNA to be distinguished from mobile proteins, offering a new perspective on protein-DNA interactions, from the molecular level to the length scale of whole bacterial cells. We demonstrate how we have applied this technique to answer key questions in transcription, chromosome organisation, and DNA repair in Escherichia coli.Discriminating transcribing RNA polymerase molecules from the rest of the population showed that low levels of transcription can occur throughout the nucleoid, but clustering analysis and 3D Structured Illumination Microscopy (SIM) showed that dense clusters of transcribing RNAPs form at the nucleoid periphery, indicating a movement of gene loci out of the bulk of DNA as levels of transcription increase [1].We further demonstrate how this technique can be used to study the formation and DNA binding of multienzyme complexes shedding light on the damage recognition mechanism of the UvrAB complex. Finally we highlight an assay to characterise the search process and non-specific DNA interactions of DNA-binding proteins by degrading the entire chromosome.1. Stracy, M. et al. Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid. Proc. Natl. Acad. Sci. 112, 201507592 (2015).

Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay.

Convenient and rapid immunofiltration assays (IFAs) enable on-site “yes” or “no” determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs)-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP). CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG) and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test.

Discrimination of "specific" and "nonspecific" binding in two-dimensional photonic crystals.

In this paper, we propose a design for a 2D slab photonic crystal (PhC) virus sensor and an associated signal analysis methodology that together enable single-virus detection while rejecting false positives that occur due to non-specific interactions of serum proteins and small molecules with the sensor surface. The slab-PhC design takes advantage of both the optical and geometrical properties of its incorporated structures by physically limiting virus infiltration to only the most sensitive region of the PhC sensor, while allowing simultaneous measurement of both site-selective virus infiltration and non-specific small molecule accumulation across the device. Notably, the proposed sensor transducer is compatible with both standard semiconductor fabrication procedures and lab-on-a-chip style microfluidic delivery systems. 3D finite-difference time-domain electromagnetic field computation results are presented, the outcomes of which indicate that both specific (target) virus capture and non-specific (non-target) binding can be simultaneously measured and discerned from one another. This type of capacity for background-corrected, single-pathogen target detection would provide a new and novel advancement toward sensitive, label-free virus diagnostics.

New potential eukaryotic substrates of the mycobacterial protein tyrosine phosphatase PtpA: hints of a bacterial modulation of macrophage bioenergetics state.

The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.

A Systematic Investigation to Determine the Optimal Lipid Coating for Nanopore-Based Sensing Experiments

Despite the importance of proteins, nanopore sensing has so far been mostly focused on single molecule DNA and RNA characterization. One factor that limited experiments with proteins were nonspecific interactions of proteins with the walls of synthetic nanopores. We showed recently, that nanopores with fluid coatings of phospholipid bilayers circumvented this problem. In addition, anchoring proteins to lipid anchors slowed down protein translocation through nanopores and enabled determination of parameters such as the shape, volume, and dipole moment of individual non-spherical proteins. To slow down the translocation time of lipid-anchored proteins further, we have recently formed highly viscous coatings from archaea-inspired lipids. These synthetic lipids are composed of two hydrophilic head groups connected by a long hydrophobic chain; each head group is also attached to an acyl chain that spans half of the membrane thickness. As these lipids contain -in one molecule- the components of a typical lipid bilayer, a single layer is sufficient to form a stable membrane. Experimentation with many lipid coatings, including a variety of archaea-inspired lipids, has revealed differences in translocation of protein, bilayer stability and event frequency. Every bilayer composition tested has been designed to optimize each of these characteristics.

Highly Viscous Coatings from Archaea-Inspired Lipids Improve Single Protein Characterization with Nanopores

Despite the importance of proteins, nanopore sensing has so far been mostly focused on single molecule DNA and RNA characterization. One factor that limited experiments with proteins were nonspecific interactions of proteins with the walls of synthetic nanopores. We showed recently, that nanopores with fluid coatings of phospholipid bilayers circumvented this problem. In addition, anchoring proteins to lipid anchors slowed down protein translocation through nanopores and enabled determination of parameters such as the shape, volume, and dipole moment of individual non-spherical proteins. To slow down the translocation time of lipid-anchored proteins further, we have recently formed highly viscous coatings from archaea-inspired lipids. These synthetic lipids are composed of two hydrophilic head groups connected by a long hydrophobic chain; each head group is also attached to an acyl chain that spans half of the membrane thickness. As these lipids contain -in one molecule- the components of a typical lipid bilayer, a single layer is sufficient to form a stable membrane. Monolayers of these new lipids have a two to twenty-fold increased viscosity than typical phospholipid bilayers. The Archaea-inspired lipids discussed here show promise in terms of slowing translocation times and the resulting data made it possible to characterize individual proteins with increased accuracy than coatings from standard phospholipid bilayers.

Dissecting the expression landscape of RNA-binding proteins in human cancers.

BackgroundRNA-binding proteins (RBPs) play important roles in cellular homeostasis by controlling gene expression at the post-transcriptional level.ResultsWe explore the expression of more than 800 RBPs in sixteen healthy human tissues and their patterns of dysregulation in cancer genomes from The Cancer Genome Atlas project. We show that genes encoding RBPs are consistently and significantly highly expressed compared with other classes of genes, including those encoding regulatory components such as transcription factors, miRNAs and long non-coding RNAs. We also demonstrate that a set of RBPs, numbering approximately 30, are strongly upregulated (SUR) across at least two-thirds of the nine cancers profiled in this study. Analysis of the protein–protein interaction network properties for the SUR and non-SUR groups of RBPs suggests that path length distributions between SUR RBPs is significantly lower than those observed for non-SUR RBPs. We further find that the mean path lengths between SUR RBPs increases in proportion to their contribution to prognostic impact. We also note that RBPs exhibiting higher variability in the extent of dysregulation across breast cancer patients have a higher number of protein–protein interactions. We propose that fluctuating RBP levels might result in an increase in non-specific protein interactions, potentially leading to changes in the functional consequences of RBP binding. Finally, we show that the expression variation of a gene within a patient group is inversely correlated with prognostic impact.ConclusionsOverall, our results provide a roadmap for understanding the impact of RBPs on cancer pathogenesis.

Proteomic Analysis of Yeast Mutant RNA Exosome Complexes

The yeast exosome is a conserved multiprotein complex essential for RNA processing and degradation. The complex is formed by a nine-subunit core that associates with two hydrolytic 3'-5' exoribonucleases. Although catalytically inert, the assembly of this nine-subunit core seems to be essential for the exosome activity, as mutations in regions that do not directly bind RNA or are not in the active sites of the exonucleases impair the function of the complex. Previously isolated mutations in the exosome core subunit Rrp43p have been shown to negatively affect the function of the complex. With the aim of investigating the effect of these mutations on the complex stability and activity, Rrp43p and its mutant forms were purified by means of the TAP method. Mass spectrometry analyses showed that lower amounts of the exosome subunits are copurified with the mutant Rrp43p proteins. Additionally, by decreasing the stability of the exosome, other nonspecific protein interactions are favored (the data have been deposited to the ProteomeXchange with identifier PXD000580). Exosome copurified with mutant Rrp43p exhibited increased exonuclease activity, suggesting higher dissociation constants for these mutant complexes. Therefore, data reported here indicate that complexes containing a mutant Rrp43p exhibit decreased stability and provide information on additional protein interactions.

Surface grafting of electrospun fibers using ATRP and RAFT for the control of biointerfacial interactions

The ability to present signalling molecules within a low fouling 3D environment that mimics the extracellular matrix is an important goal for a range of biomedical applications, both in vitro and in vivo. Cell responses can be triggered by non-specific protein interactions occurring on the surface of a biomaterial, which is an undesirable process when studying specific receptor-ligand interactions. It is therefore useful to present specific ligands of interest to cell surface receptors in a 3D environment that minimizes non-specific interactions with biomolecules, such as proteins. In this study, surface-initiated atom transfer radical polymerization (SI-ATRP) of poly(ethylene glycol)-based monomers was carried out from the surface of electrospun fibers composed of a styrene/vinylbenzyl chloride copolymer. Surface initiated radical addition-fragmentation chain transfer (SI-RAFT) polymerisation was also carried out to generate bottle brush copolymer coatings consisting of poly(acrylic acid) and poly(acrylamide). These were grown from surface trithiocarbonate groups generated from the chloromethyl styrene moieties existing in the original synthesised polymer. XPS was used to characterise the surface composition of the fibers after grafting and after coupling with fluorine functional XPS labels. Bottle brush type coatings were able to be produced by ATRP which consisted of poly(ethylene glycol) methacrylate and a terminal alkyne-functionalised monomer. The ATRP coatings showed reduced non-specific protein adsorption, as a result of effective PEG incorporation and pendant alkynes groups existing as part of the brushes allowed for further conjugation of via azide-alkyne Huisgen 1,3-dipolar cycloaddition. In the case of RAFT, carboxylic acid moieties were effectively coupled to an amine label via amide bond formation. In each case XPS analysis demonstrated that covalent immobilisation had effectively taken place. Overall, the studies presented an effective platform for the preparation of 3D scaffolds which contain effective conjugation sites for attachment of specific bioactive signals of interest, as well as actively reducing non-specific protein interactions.

Improving the spectral analysis of fluorescence resonance energy transfer in live cells: Application to interferon receptors and Janus kinases

The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.

Conservation of Functional Sites on Interleukin-6 and Implications for Evolution of Signaling Complex Assembly and Therapeutic Intervention

A number of secreted cytokines, such as interleukin-6 (IL-6), are attractive targets for the treatment of inflammatory diseases. We have determined the solution structure of mouse IL-6 to assess the functional significance of apparent differences in the receptor interaction sites (IL-6Rα and gp130) suggested by the fairly low degree of sequence similarity with human IL-6. Structure-based sequence alignment of mouse IL-6 and human IL-6 revealed surprising differences in the conservation of the two distinct gp130 binding sites (IIa and IIIa), which suggests a primacy for site III-mediated interactions in driving initial assembly of the IL-6/IL-6Rα/gp130 ternary complex. This is further supported by a series of direct binding experiments, which clearly demonstrate a high affinity IL-6/IL-6Rα-gp130 interaction via site III but only weak binding via site II. Collectively, our findings suggest a pathway for the evolution of the hexameric, IL-6/IL-6Rα/gp130 signaling complex and strategies for therapeutic targeting. We propose that the signaling complex originally involved specific interactions between IL-6 and IL-6Rα (site I) and between the D1 domain of gp130 and IL-6/IL-6Rα (site III), with the later inclusion of interactions between the D2 and D3 domains of gp130 and IL-6/IL-6Rα (site II) through serendipity. It seems likely that IL-6 signaling benefited from the evolution of a multipurpose, nonspecific protein interaction surface on gp130, now known as the cytokine binding homology region (site II contact surface), which fortuitously contributes to stabilization of the IL-6/IL-6Rα/gp130 signaling complex.

Method for suppressing non-specific protein interactions observed with affinity resins

Previous high throughput data analysis from several different approaches to affinity purification of protein complexes have revealed catalogues of contaminating proteins that persistently co-purify. Some of these contaminating proteins appear to be specific to one particular affinity matrix used or even to the artificial affinity tags introduced into endogenous proteins for the purpose of purification.A recent approach to minimising non-specific protein interactions in high throughput screens utilises pre-equilibration of affinity surfaces with thiocyanate anions to reduce non-specific binding of proteins. This approach not only reduces the effect of contaminating proteins but also promotes the enrichment of the specific binding partners. Here, we have taken this method and adapted it in an attempt to reduce the abundance of common contaminants in affinity purification experiments. We found the effect varied depending on the bait used, most likely due to its endogenous abundance.

A computerized protein–protein interaction modeling study of ampicillin antibody specificity in relation to biosensor development

Nonspecific interactions between immobilized biomolecules and interfering proteins significantly impede biosensor development and commercialization. Advances in bioinformatics and computer technology have facilitated a greater understanding of biological interactions. We employed two different protein-protein docking programs to simulate the nonspecific interaction between ampicillin antibody and potential interfering proteins (human serum albumin and ovalbumin). To evaluate the contact and probability of association with the active site of the antibody, different amino acid chains from human serum albumin (HSA) and ovalbumin (OVA) were modeled in the simulation. In addition, a well-known specific immune complex, lysozyme and lysozyme antibody, was simulated for comparison. The results demonstrated that the cluster density of nonspecific interactions was smaller than the specific interaction between lysozyme and antibody, and that the dock scores were scattered. However, the active site of ampicillin antibody was prone to nonspecific protein interactions. The strength of interaction was different for specific binding and nonspecific binding. These results provide a platform for detecting the probability of nonspecific interactions and for improving methods of biosensor detection construction with reduced nonspecific adsorption.

Structural basis for site-specific ribose methylation by box C/D RNA protein complexes

Box C/D RNA protein complexes (RNPs) direct site-specific 2'-O-methylation of RNA and ribosome assembly. The guide RNA in C/D RNP forms base pairs with complementary substrates and selects the modification site using a molecular ruler. Despite many studies of C/D RNP structure, the fundamental questions of how C/D RNAs assemble into RNPs and how they guide modification remain unresolved. Here we report the crystal structure of an entire catalytically active archaeal C/D RNP consisting of a bipartite C/D RNA associated with two substrates and two copies each of Nop5, L7Ae and fibrillarin at 3.15-Å resolution. The substrate pairs with the second through the eleventh nucleotide of the 12-nucleotide guide, and the resultant duplex is bracketed in a channel with flexible ends. The methyltransferase fibrillarin binds to an undistorted A-form structure of the guide-substrate duplex and specifically loads the target ribose into the active site. Because interaction with the RNA duplex alone does not determine the site specificity, fibrillarin is further positioned by non-specific and specific protein interactions. Compared with the structure of the inactive C/D RNP, extensive domain movements are induced by substrate loading. Our results reveal the organization of a monomeric C/D RNP and the mechanism underlying its site-specific methylation activity.

Anti-fouling bioactive surfaces

Bioactive surfaces refer to surfaces with immobilized bioactive molecules aimed specifically at promoting or supporting particular interactions. Such surfaces are of great importance for various biomedical and biomaterials applications. In the past few years, considerable effort has been made to create bioactive surfaces by forming specific biomolecule-modified surfaces on a non-biofouling “base” or “background”. Hydrophilic and bioinert polymers have been widely used as anti-fouling layers that resist non-specific protein interactions. They can also serve as “spacers” to effectively move the immobilized biomolecule away from the surface, thus enhancing its bioactivity. In this review we summarize several successful approaches for the design and preparation of bioactive surfaces based on different types of anti-fouling/spacer materials. Some perspectives on future research in this area are also presented.