Intrinsic Affinity Research Articles

Water Structure Recovery in Chaotropic Anion Recognition: High-Affinity Binding of Dodecaborate Clusters to γ-Cyclodextrin.

Dodecaborate anions of the type B12X122− and B12X11Y2− (X=H, Cl, Br, I and Y=OH, SH, NH3+, NR3+) form strong (Ka up to 106 L mol−1, for B12Br122−) inclusion complexes with γ-cyclodextrin (γ-CD). The micromolar affinities reached are the highest known for this native CD. The complexation exhibits highly negative enthalpies (up to −25 kcal mol−1) and entropies (TΔS up to −18.4 kcal mol−1, both for B12I122−), which position these guests at the bottom end of the well-known enthalpy-entropy correlation for CDs. The high driving force can be traced back to a chaotropic effect, according to which chaotropic anions have an intrinsic affinity to hydrophobic cavities in aqueous solution. In line with this argument, salting-in effects revealed dodecaborates as superchaotropic dianions.

Revisiting the Dragon Princess: Her Role in Medieval Engi Stories and Their Implications in Reading the Lotus Sutra

This article examines how the Dragon Princess, one of the most celebrated characters in the Lotus Sutra, is represented in the noh drama Ama and the Heike Nokyo sutra set. By doing so, it debunks the prevailing consensus in understanding the Dragon Princess and her episode in the sutra, and illustrates a hitherto unnoticed intrinsic affinity between medieval Japanese engi stories and Buddhist scriptural narratives.KEYWORDS: Lotus Sutra-Dragon Princess-henjo nanshi-ryunyo jobutsu- Buddhism-gender and narrativity-Heike nokyo-ama (female diver)(ProQuest: ... denotes non-US-ASCII text omitted.)The Heike n?ky?, a twelfth-century set of thirty-three scriptural scrolls preserved at Itsukushima Shrine in Hiroshima Prefecture, is one of the most sumptuous and elaborate examples of medieval Japanese illustrated Buddhist scriptural manuscripts. One of the scrolls, the "Devadatta" chapter of the Lotus Sutra, bears a frontispiece that features the Dragon Princess, the celebrated protagonist of the chapter (figure 1). The illustration is arguably the most sublime representation of the princess in visual art. Although recent studies of the Dragon Princess have made her famous for her sudden change of sex and transformation into a Buddha, the princess in the frontispiece simply offers her jewel to the Buddha. She shows no sign of physical metamorphosis. What sort of interpretive strategies are required to understand her act as represented in the frontispiece?Engi Literature and Buddhist Scriptural NarrativesThis article aims at illustrating an intrinsic affinity between medieval Japanese engi ... materials in textual, performative, and visual genres, and Buddhist scriptural materials. The prevailing consensus among scholars has long held that the term engi is an appropriation of the Buddhist doctrinal term "dependent co-origination" (Sk. prat?tyasa?utp?da), which is translated in the Chinese canon as innen sh?ki ... (Ch. yinyuan shengqi) and abbreviated in more common use as engi (Ch. yuanqi). However, the concept of dependent coorigination means that all things in the world are intrinsically neither rising nor ceasing; everything originates from its non-origin. Obviously such a philosophical idea would not accord well with medieval engi narratives, which are designed to account for the specific provenance of temples, shrines, or other sacred sites, the divinities worshipped there, and/or visual and material representations of those divinities.Although it is not as well known today, an alternative etymology links the word engi to another significant Buddhist term, namely, nid?na (Jp. nidana; Ch. nituona ...), which literally means origin (sho ....), provenance (hokki ...), cause (in ...), and original cause (shoin ...). More importantly for this article, nid?na is the name for one of the twelve categories of Buddhist scriptural discourse (Sk. dv?da???ga-dharma-pravacana; Ch. shierbujing; Jp. j?nibuky? ... or ...). Nid?na in this sense is translated more commonly as either engi or innen ... As a category of scriptural narratives, nid?na or engi consist of stories that aim at revealing the original cause of sermons delivered by the Buddha, vinaya rules he established, and strange and miraculous events caused or witnessed by him. In most cases, the origins of the events described are found in the former lives of the Buddha or his disciples in the distant past. In that regard, nid?na are closely linked to two other narrative categories in scriptural discourse, namely, j?taka (Ch. bensheng; Jp. honj? ...), stories of the Buddha's former lives; and itiv?ttaka (Ch. benshi; Jp. honji ...), stories of the former lives of the Buddha's disciples, both ordained and lay.1Because nid?na, j?taka, and itiv?ttaka are often employed as elaborate, didactic narrative figures within the scriptures, they often overlap with avad?na (Ch. piyu; Jp. hiyu ...), yet another narrative entity within the twelve categories, which consists of teaching by means of metaphors and parables. …

Affinity for self drives the preferential accumulation of promiscuous CD4+ T cells over the lifespan. (LYM2P.726)

Abstract T cells discriminate self from foreign peptides presented in the context of self-major histocompatibility complex (pMHC) molecules via clonotypic T cell receptors (TCRs). CD8+ T cell recognition and responsiveness to foreign pMHC is known to diminish over the lifespan, which is consistent with gradual thymic involution over time. How the affinity of the CD4+ T cell compartment for self-pMHC, and its capacity to bind foreign-pMHC change over the lifespan are basic aspects of T cell biology that remain largely unexplored. Experimentally restricting thymic selection is known to allow degenerate CD4+ T cells to develop. This suggests that they might accumulate in the CD4+ T cell compartment over time. We report that, while old mice (18-22 months) contain fewer CD4+ T cells than adults (8-12 weeks), those that remain have a higher intrinsic affinity for self-pMHC. Old mice also have more cells that bind distinct foreign-pMHCs, either alone or in combination. The numerical increase of these subsets with age directly correlates with their affinity for self-pMHC. However, no relationship was observed between affinity for self-pMHC and responsiveness to foreign-pMHC. These data demonstrate that the CD4+ T cell compartment preferentially accumulates promiscuous constituents with age as a consequence of higher affinity T cell receptor interactions with self-pMHC. These results have important implications for the design of immunotherapeutics targeting CD4+ T cells for long-term efficacy.

Identification of RNA cargoes by antibody-positioned RNA amplification.

The interactions between various RNA-binding proteins (RBPs) and the RNA transcripts they bind strongly influence posttranscriptional control of gene expression in vertebrates. The hundreds of vertebrate RBPs that have been identified within the genome, often with multiple RNA recognition motifs, are capable of recognizing specific target RNA sequences mediating the maturation, movement, and translational state of their RNA cargoes. To identify the cargoes associated with a specific RBP, we have developed a technique called antibody-positioned RNA amplification (APRA), which positions an oligonucleotide with a degenerate priming sequence in proximity to the RNAs sequestered by a specific RBP. The conjugation of the priming oligonucleotide to the antibody by itself does not interfere with the antibody's intrinsic affinity for the target RBP epitope, thus enabling RNA targets to be reverse-transcribed and amplified via a T7 bacteriophage RNA polymerase promoter sequence located upstream of the degenerate priming sequence in the oligonucleotide. By identifying the mRNA transcripts associated with the RBP in situ, we may be able to ascertain the significance of their temporal expression and physiological activities within the vast transcriptional networks regulating functional responses to stimuli.

Outdoor Performance and Stability under Elevated Temperatures and Long‐Term Light Soaking of Triple‐Layer Mesoporous Perovskite Photovoltaics

AbstractLack of proven stability has become a major obstacle on the path of metal halide perovskite solar cells (PSCs), in particular methylammonium lead triiodide (MAPbI3), towards commercial viability. This correlates with the intrinsic affinity of MAPbI3 towards moisture and ambient air in particular, leading to its degradation in ambient conditions. We performed extensive stability tests to prove the durability of hole‐conductor‐free PSCs based on a triple‐layer architecture employing carbon as a back contact, including outdoor tests in the hot desert climate and indoor long‐term light soaking as well as heat exposure during 3 months at 80–85 °C. These results show no evidence for device degradation under the test conditions, confirming that the triple‐layer device architecture provides a promising path towards realizing efficient and stable perovskite photovoltaics.

Driving force-dependent block by internal Ba2+ on the Kir2.1 channel: Mechanistic insight into inward rectification

The Kir2.1 channel is characterized by strong inward rectification; however, the mechanism of the steep voltage dependence near the equilibrium potential remains to be investigated. Here, we studied the internal Ba2+ block of the Kir2.1 channel expressed in Xenopus oocytes. We showed that the driving force and thus the K+ ion flux significantly influenced the apparent affinity of the block by internal Ba2+. Kinetic analysis revealed that the binding rate shifted with the driving force and changed steeply near the equilibrium point, either in the presence or absence of the transmembrane electrical field. The unbinding rate was determined by the intrinsic affinity of the site. Mutagenesis studies revealed that the high-affinity binding site for Ba2+ was located near T141 at the internal entrance of the selectivity filter. The steep change of the blocking affinity near the equilibrium potential may result from the flux-coupling effect in the single-file, multi-ion cytoplasmic pore.

Oligomerization of the Polycystin-2 C-terminal Tail and Effects on Its Ca2+-binding Properties

Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca(2+)-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca(2+)-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca(2+) bound, although TRP channels are believed to be tetramers. We found that there is only one Ca(2+)-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca(2+) binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca(2+) binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca(2+)-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca(2+) binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca(2+)-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca(2+)-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca(2+)-binding proteins.

Preparation of abiotic polymer nanoparticles for sequestration and neutralization of a target peptide toxin.

Synthetic polymer nanoparticles (NPs) with intrinsic affinity for target biomacromolecules hold great promise in the development of novel tools for biological and biomedical research. We recently reported the design and synthesis of abiotic, synthetic polymer NPs with high intrinsic affinity for a peptide toxin melittin. The NP was selected by screening a small library of NPs (∼100 nm) composed of various ratios of monomers that contain functional groups complementary to the peptide melittin. The selected polymer NP, a co-polymer of acrylic acid (AAc), N-tert-butylacrylamide (TBAm), N-isopropylacrylamide (NIPAm) and N,N'-methylenebisacrylamide (BIS), effectively captures and neutralizes the toxicity of the peptide through a combination of electrostatic and hydrophobic interactions. This protocol describes a step-by-step procedure for the preparation and evaluation of synthetic polymer NPs for sequestration and neutralization of the target peptide toxin. The polymer NPs can be synthesized in a one-step polymerization reaction using commercially available reagents. The polymerization reaction for the synthesis of polymer NPs takes several hours, and the total protocol including subsequent purification and characterization by dynamic light scattering, NMR and toxicity neutralization assays takes 1-2 weeks in total.

Affinity and Selectivity of Peptides for Inorganic Materials: A Thermodynamic Discussion of the Role of Conformational Flexibility

The recognition of inorganic materials by polypeptides is a technologically relevant and scientifically intriguing phenomenon. Several features of the polypeptide determine the affinity for a given surface and, possibly, its selectivity among a set of materials or among various crystal planes of the same material. Different amino acids have different intrinsic affinities for a given surface; the geometrical features of the surface are also relevant in the recognition process. By means of a minimal, analytically solvable thermodynamic model for the polypeptide-surface interaction, we discuss the role of the conformational flexibility in determining affinity and selectivity of polypeptides for surfaces. In particular, the interplay between geometrical matching of polypeptide-surface features and the presence in the polypeptide of amino acids able to bind the target surface is analyzed as a function of the flexibility. A discussion of literature data in the light of this minimal model is also given.

HDL-mimetic PLGA nanoparticle to target atherosclerosis plaque macrophages.

High-density lipoprotein (HDL) is a natural nanoparticle that exhibits an intrinsic affinity for atherosclerotic plaque macrophages. Its natural targeting capability as well as the option to incorporate lipophilic payloads, e.g., imaging or therapeutic components, in both the hydrophobic core and the phospholipid corona make the HDL platform an attractive nanocarrier. To realize controlled release properties, we developed a hybrid polymer/HDL nanoparticle composed of a lipid/apolipoprotein coating that encapsulates a poly(lactic-co-glycolic acid) (PLGA) core. This novel HDL-like nanoparticle (PLGA-HDL) displayed natural HDL characteristics, including preferential uptake by macrophages and a good cholesterol efflux capacity, combined with a typical PLGA nanoparticle slow release profile. In vivo studies carried out with an ApoE knockout mouse model of atherosclerosis showed clear accumulation of PLGA-HDL nanoparticles in atherosclerotic plaques, which colocalized with plaque macrophages. This biomimetic platform integrates the targeting capacity of HDL biomimetic nanoparticles with the characteristic versatility of PLGA-based nanocarriers.

Role of the Transmembrane α-Helix M4 in the Potentiation of Pentameric Ligand-Gated Ion Channels

The gating of pentameric ligand gated ion channels (pLGICs) is sensitive to a variety of allosteric modulators that act on the transmembrane domain, including lipids. Here, we use two prokaryotic homologs, GLIC and ELIC, to examine the role of the lipid-exposed transmembrane a-helix, M4, in the allosteric regulation of pLGICs. Aromatic interactions at the interface between M4 and the adjacent a-helices, M1 and M3, drive M4 binding during folding (Haeger et al. (2010) Nat Struct Mol Biol 17:90-98). Alanine substitutions of the aromatics at this interface in GLIC weaken M4 binding and inhibit channel function, while aromatic substitutions of aliphatic residues at the same interface in ELIC promote M4 binding and potentiate channel function. The strength of M4 binding to M1/M3 governs the susceptibility of both pLGICs to the potentiating effects of a congenital myasthenic syndrome mutation, which occurs in muscle-type nicotinic receptors on the lipid-facing surface of M4 and alters lipid-protein interactions to enhance function, but has little effect on the inhibitory activity of the drug, propofol. We thus identify both M4-dependent and M4-independent pathways for transmembrane domain allosteric regulation. Our data suggest that the chemistry at the interface between M4 and M1/M3 influences the intrinsic M4 binding affinity for M1/M3 and may thus govern the susceptibility of a pLGIC to the potentiating effects of lipids.

Characterization of PC2 Cterm Calcium-Binding Interaction and its Structural Implications

Polycystin-2 (PC2) is a Ca2+-regulated Ca2+-channel from the transient receptor potential (TRP) family. Mutations in PC2 can cause autosomal dominant polycystic kidney disease (ADPKD). The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is crucial for channel assembly and function. We have combined biophysical and structural approaches to characterize the Ca2+-dependent molecular mechanism within the C-terminal tail that is involved in channel assembly and functional regulation. We have determined that HPC2 Cterm forms a trimer in solution with and without Ca2+-bound, even though TRP channels are generally tetrameric. We have definitively shown that there is only one Ca2+-binding site in HPC2 Cterm, located within its EF-hand domain. However, its Ca2+-binding affinity is greatly enhanced relative to its intrinsic binding affinity in the isolated EF-hand, possibly due to the positive cooperativity from the trimer interaction. We also employed sea urchin PC2 as a parallel model to study. The sea urchin C-terminal domain construct (SUPC2 Ccore) also trimerizes in solution independent of Ca2+-binding. In contrast, the SUPC2 Ccore contains two Ca2+-binding sites within its EF-hand domain, which exhibit cooperative Ca2+-binding due to internal stabilization. Consequently, trimerization does not further improve Ca2+-binding affinity in SUPC2 Ccore relative to the isolated EF-hand domain. Using both hydrogen-deuterium exchange mass spectroscopy and nuclear magnetic resonance, we have localized the Ca2+-binding sites in PC2 C-terminal tail and mapped the conformational changes induced by Ca2+-binding. We demonstrate that in addition to the direct, local stabilizing effects within the EF-hand, Ca2+-binding also causes conformational changes in the distal coiled-coil domain. This study provides a structural basis for regulation of the PC2 channel by its cytosolic C-terminal domain, with an improved understanding of the functional role of PC2 in regulating intracellular Ca2+ signaling.

Yeast GSK-3 kinase regulates astral microtubule function through phosphorylation of the microtubule-stabilizing kinesin Kip2

ABSTRACTThe S. cerevisiae kinesin Kip2 stabilises astral microtubules (MTs) and facilitates spindle positioning through transport of MT-associated proteins, such as the yeast CLIP-170 homologue Bik1, dynein and the adenomatous-polyposis-coli-related protein Kar9 to the plus ends of astral MTs. Here, we show that Kip2 associates with its processivity factor Bim1, the yeast homologue of the plus-end-tracking protein EB1. This interaction requires an EB1-binding motif in the N-terminal extension of Kip2 and is negatively regulated by phosphorylation through Mck1, the yeast glycogen synthase kinase 3. In addition, Mck1-dependent phosphorylation decreases the intrinsic MT affinity of Kip2. Reduction in Kip2 phosphorylation leads to stabilisation of astral MTs, and accumulation of Kip2, dynein and Kar9 at MT plus ends, whereas loss of Mck1 function leads to defects in spindle positioning. Furthermore, we provide evidence that a subpopulation of Mck1 at the bud-cortex phosphorylates Kip2. We propose that yeast GSK-3 spatially controls astral MT dynamics and the loading of dynein and Kar9 on astral MT plus ends by regulating Kip2 interactions with Bim1 and MTs.

The N-Terminal Hypervariable Region of Troponin T Differentially Modulates the Affinity of Tropomyosin-Binding Sites

The troponin complex plays a central role in the allosteric function of sarcomeric thin filaments by enacting conformational changes during the Ca2+-regulated contraction and relaxation of striated muscle. The troponin subunit T (TnT) has two binding sites for tropomyosin (Tm) and is responsible for anchoring the troponin complex to the thin filament. Although the C-terminal and middle regions of the TnT polypeptide chain are highly conserved among the three muscle type isoforms, the hypervariable N-terminal region has evolutionarily diverged significantly among isoforms. Previous studies have shown that the N-terminal variable region fine-tunes Ca2+ regulation of muscle contractility via modulation of the overall molecular conformation of TnT, and its interactions with Tm. In the present study, we engineered intact TnT and representative fragments of TnT, expressed them in E. coli, and prepared purified proteins for functional studies. Tropomyosin binding affinity was analyzed using affinity chromatography and solid phase protein binding assays to investigate the modulatory effects of the N-terminal variable region. The results demonstrated that in the absence of the N-terminal variable region, TnT's conserved middle region and C-terminal T2 region Tm-binding sites showed comparable Tm-binding affinities across isoforms. The data demonstrate that without the modulatory effect of the N-terminal variable region, the intrinsic Tm-binding affinities of the two sites are both high. In contrast, the presence of the isoform specific N-terminal variable region differentially reduces the binding affinity of TnT for Tm, primarily at the middle region binding site. These novel findings indicate that the N-terminal variable region plays a key role in the functional difference of muscle fiber type-specific, developmental, splice variant, and pathogenic TnT isoforms by modulating the interactions with Tm during the contraction and relaxation of cardiac and skeletal muscle.

Metal loading of lanthanidopolymers driven by positive cooperativity.

This work demonstrates how the thermodynamic loading of monodisperse polymeric single-stranded multi-tridentate receptors of variable lengths is controlled by the nature of the metallic carrier Ln(hfac)3 (Ln is La, Eu or Y, and hfac is hexafluoroacetylacetonate). Whereas the intrinsic affinity of the tridentate binding site is maximum for medium-sized Eu(3+) and decreases for Y(3+), the contraction of the hydrodynamic radius of the polymer upon metal loading induces positive allosteric cooperativity for the smaller cations. The origin of this behaviour is rationalized within the frame of intermetallic dipole-dipole interactions modulated by the solvation potential of dipolar solutes in dielectric materials. Positive cooperativity produces local high-density of metal ions along the ligand strands (metal clustering) with potential interest in energy migration and sensing processes.

Exploring the correlation between the sequence composition of the nucleotide binding G5 loop of the FeoB GTPase domain (NFeoB) and intrinsic rate of GDP release.

GDP release from GTPases is usually extremely slow and is in general assisted by external factors, such as association with guanine exchange factors or membrane-embedded GPCRs (G protein-coupled receptors), which accelerate the release of GDP by several orders of magnitude. Intrinsic factors can also play a significant role; a single amino acid substitution in one of the guanine nucleotide recognition motifs, G5, results in a drastically altered GDP release rate, indicating that the sequence composition of this motif plays an important role in spontaneous GDP release. In the present study, we used the GTPase domain from EcNFeoB (Escherichia coli FeoB) as a model and applied biochemical and structural approaches to evaluate the role of all the individual residues in the G5 loop. Our study confirms that several of the residues in the G5 motif have an important role in the intrinsic affinity and release of GDP. In particular, a T151A mutant (third residue of the G5 loop) leads to a reduced nucleotide affinity and provokes a drastically accelerated dissociation of GDP.

Modulating the physicochemical and structural properties of gold-functionalized protein nanotubes through thiol surface modification.

Biomolecules are advantageous scaffolds for the synthesis and ordering of metallic nanoparticles. Rotavirus VP6 nanotubes possess intrinsic affinity to metal ions, a property that has been exploited to synthesize gold nanoparticles over them. The resulting nanobiomaterials have unique properties useful for novel applications. However, the formed nanobiomaterials lack of colloidal stability and flocculate, limiting their functionality. Here we demonstrate that it is possible to synthesize thiol-protected gold nanoparticles over VP6 nanotubes, which resulted in soluble nanobiomaterials. With this strategy, it was possible to modulate the size, colloidal stability, and surface plasmon resonance of the synthesized nanoparticles by controlling the content of the thiolated ligands. Two types of water-soluble ligands were tested, a small linear ligand, sodium 3-mercapto-1-propanesulfonate (MPS), and a bulky ligand, 5-mercaptopentyl β-D-glucopyranoside (GlcC5SH). The synthesized nanobiomaterials had a higher stability in suspension, as determined by Z-potential measurements. To the extent of our knowledge, this is the first time that a rational strategy is developed to modulate the particular properties of metal nanoparticles in situ synthesized over a protein bioscaffold through thiol coating, achieving a high spatial and structural organization of nanoparticles in a single integrative hybrid structure.

Effect of phloretin on the binding of 1-anilino-8-naphtalene sulfonate (ANS) to 1,2-Dimyristoyl-sn-glycero-3-phosphocoline (DMPC) vesicles in the gel and liquid-crystalline state.

Phloretin is a known modifier of the internal dipole potential of lipid membranes. We studied the interaction of phloretin with model lipid membranes and how it influences the membrane dipole organization using ANS as fluorescent probe. The fluorescence increase observed when ANS binds to DMPC liposomes in gel phase (13 °C) was 2.5 times larger in the presence of phloretin. This effect was due to an increase in ANS affinity, which can be related to the known capability of phloretin in decreasing the dipole potential. Conversely, when the experiments were carried out at 33 °C (liquid crystalline phase), phloretin completely inhibited the increase in ANS fluorescence. In addition, phloretin only affected the electrical properties of the membrane in the gel phase, whereas it modifies structural ones in the liquid-crystalline state. We postulate that phloretin was bound only to the DMPC interface in the gel phase decreasing the surface negative charge density without modifying the structural properties of the ANS binding sites. In the liquid-crystalline phase instead, it increased the accessibility of water to the ANS binding sites decreasing the intrinsic affinity and the fluorescence quantum yield of ANS.

Bi-epitope SPR surfaces: a solution to develop robust immunoassays.

Surface plasmon resonance (SPR)-based immunoassays have numerous applications and require high affinity reagents for sensitive and reliable measurements. We describe a quick approach to turn low affinity antibodies into appropriate capture reagents. We used antibodies recognizing human ephrin type A receptor 2 (EphA2) and a ProteOn XPR36 as a model system. We generated so-called ‘bi-epitope’ sensor surfaces by immobilizing various pairs of anti-EphA2 antibodies using standard amine coupling. The apparent binding affinities to EphA2 and EphA2 detection sensitivities of the bi-epitope and ‘single-epitope’ surfaces were then compared. For all antibody pairs tested, bi-epitope surfaces exhibited an ∼10–100-fold improvement in apparent binding affinities when compared with single-epitope ones. When pairing 2 antibodies of low intrinsic binding affinities (∼10−8 M) and fast dissociation rates (∼10−2 s−1), the apparent binding affinity and dissociation rate of the bi-epitope surface was improved up to ∼10–10 M and 10−4 s−1, respectively. This led to an ∼100–200-fold enhancement in EphA2 limit of detection in crude cell supernatants. Our results show that the use of antibody mixtures in SPR applications constitutes a powerful approach to develop sensitive immunoassays, as previously shown for non-SPR formats. As SPR-based assays have significantly expanded their reach in the last decade, such an approach promises to further accelerate their development.

Affinities of human histo-blood group antigens for norovirus capsid protein complexes.

The binding profiles of many human noroviruses (huNoVs) for human histo-blood group antigens have been characterized. However, quantitative-binding data for these important virus-host interactions are lacking. Here, we report on the intrinsic (per binding site) affinities of HBGA oligosaccharides for the huNoV VA387 virus-like particles (VLPs) and the associated subviral P particles measured using electrospray ionization mass spectrometry. The affinities of 13 HBGA oligosaccharides, containing A, B and H epitopes, with variable sizes (disaccharide to tetrasaccharide) and different precursor chain types (types 1, 2, 3, 5 and 6), were measured for the P particle, while the affinities of the A and B trisaccharides and A and B type 6 tetrasaccharides for the VLP were determined. The intrinsic affinities of the HBGA oligosaccharides for the P particle range from 500 to 2300 M(-1), while those of the A and B trisaccharides and the A and B type 6 tetrasaccharides for the VLP range from 1000 to 4000 M(-1). Comparison of these binding data with those measured previously for the corresponding P dimer reveals that the HBGA oligosaccharides tested exhibit similar intrinsic affinities for the P dimer and P particle. The intrinsic affinities for the VLP are consistently higher than those measured for the P particle, but within a factor of three. While the cause of the subtle differences in HBGA oligosaccharide affinities for the P dimer and P particle and those for the VLP remains unknown, the present data support the use of P dimers or P particles as surrogates to the VLP for huNoV-receptor-binding studies.