A fractal analysis of analyte-estrogen receptor binding and dissociation kinetics using biosensors: environmental and biomedical effects

A fractal analysis is used to model the binding and dissociation kinetics between analytes in solution and estrogen receptors (ERs) immobilized on a sensor chip of a surface plasmon resonance (SPR) biosensor. The influence of different ligands is also analyzed. A better understanding of the kinetics provides physical insights into the interactions, and suggests means by which appropriate interactions (to promote correct signaling) and inappropriate interactions such as with xenoestrogens (to minimize inappropriate and deleterious to health signaling) may be better controlled. The fractal approach is applied to analyte–ER interaction data available in the literature. The units for the different parameters (rate coefficients and affinities) in fractal-type kinetics are different from those obtained in classical kinetics. Numerical values obtained for the binding and the dissociation rate coefficients are linked to the degree of roughness or heterogeneity (fractal dimension, D_f) present on the biosensor chip surface. In general, the binding and the dissociation rate coefficients are very sensitive to the degree of heterogeneity on the surface. A single-fractal analysis is adequate in some cases. In others (that exhibit complexities in the binding or the dissociation curves) a dual-fractal analysis is required to obtain a better fit. This has biomedical and environmental implications in that the dissociation (and the binding) rate coefficient may be used to alleviate (deleterious effects) or enhance (beneficial effects) by selective modulation of the surface. The affinity values obtained in the analysis are consistent with the numbers required to (a) promote signaling between the correct analyte and the estrogen receptor, and (b) minimize the signaling between xenoestrogens and the estrogen receptor.     

Analysis of Cre-loxP interaction by surface plasmon resonance: influence of spermidine on cooperativity

To study target site selectivity of one important class of DNA-binding proteins, site-specific DNA recombinases, we developed an automated real-time kinetic assay based on surface plasmon resonance (BIACORE) and formulated a curve-fitting model that takes into account cooperative interactions. Monitoring the interaction between the Cre DNA recombinase and its specific target site loxP by BIACORE, we found that Cre associates with loxP tightly and highly cooperatively. We observed that the cooperative moment of the Cre-loxP interaction is strongly dependent on the concentration of spermidine, a small polyamine influencing DNA conformation. Thus, DNA conformation can have a profound impact on substrate recognition and subsequent recombination.     

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

A new approach for analyzing lipid-lipid transfer protein interactions is described. The transfer protein is genetically engineered for expression with a C-terminal biotinylated peptide extension (AviTag). This allows protein anchoring to a streptavidin-coated chip for surface plasmon resonance (SPR)-based assessment of lipid binding. Sterol carrier protein-2 (SCP-2), involved in the intracellular trafficking of cholesterol, fatty acids, and other lipids, was selected as the prototype. Biotinylated SCP-2 (bSCP-2) was expressed in Escherichia coli, purified to homogeneity by mutated streptavidin (SoftLink) affinity chromatography, and confirmed by mass spectrometry to contain one biotin group at the expected position. Intermembrane [(14)C]cholesterol transfer was strongly enhanced by bSCP-2, demonstrating that it was functional. Using bSCP-2 immobilized on a Biacore streptavidin chip, we determined on- and off-rate constants along with equilibrium dissociation constants for the following analytes: oleic acid, linoleic acid, cholesterol, and fluorophore (NBD)-derivatized cholesterol. The dissociation constant for NBD-cholesterol was similar to that determined by fluorescence titration for SCP-2 in solution, thereby validating the SPR approach. This method can be readily adapted to other transfer proteins and has several advantages over existing techniques for measuring lipid binding, including (i) the ability to study lipids in their natural states (i.e., without relatively large reporter groups) and (ii) the ability to measure on- and off- rate constants as well as equilibrium constants.     

Quantitative analysis of EGFR affinity to immobilized glycolipids by surface plasmon resonance

EGF-induced activation of EGFR tyrosine kinase is known to be inhibited by ganglioside GM3, its dimer, and other mimetics. However, details of the interaction, such as kinetic properties, have not yet been clarified. The direct interaction is now defined by the surface plasmon resonance (SPR) technique. To determine the affinity of EGFR for lyso-GM3 or lyso-GM3 mimetic, these glycolipid ligands were covalently immobilized onto a sensor chip, and binding affinities were investigated. Results of these studies confirmed the direct interaction of lyso-GM3 or its mimetic with EGFR. A strong interaction between EGFR and lyso-GM3 or its mimetic was indicated by increased binding of EGFR to glycolipid-immobilized surface, in an EGFR dose-dependent manner.     

A mathematical analysis using fractals for binding interactions of nuclear estrogen receptors occurring on biosensor surfaces

A mathematical approach using fractal concepts is presented for modeling the binding and dissociation interactions between analytes and nuclear estrogen receptors (ER) occurring on surface plasmon resonance biosensor chip surfaces. A kinetic knowledge of the binding interactions mediated by ER would help in better understanding the carcinogenicity of these steroidogenic compounds and assist in modulating these reactions. The fractal approach is applied to analyte-ER interaction data obtained from literature. Numerical values obtained for the binding and dissociation rate coefficients are linked to the degree of roughness or heterogeneity (fractal dimension, D(f)) present on the biosensor surface. For example, a single-fractal analysis is used to describe the binding and dissociation phases for the binding of estradiol and ERalpha in solution to clone 31 protein immobilized on a biosensor chip (C-S. Suen et al., 1998, J. Biol. Chem. 273(42), 27645-27653). The binding and the dissociation rate coefficients are 27.57 and 8.813, respectively, and the corresponding fractal dimensions are 1.986 and 2.268, respectively. In some examples dual-fractal models were employed to obtain a better fit of either the association or the dissociation phases or for both. Predictive relationships are developed for (a) the binding and the dissociation rate coefficients as a function of their respective fractal dimensions and (b) the ratio K(A) (= k/k(d)) as a function of the ratio of the fractal dimensions (D(f)/D(fd)). The analysis should provide further physical insights into the ER-mediated interactions occurring on biosensor and other surfaces.     

Characterization of the interactions between Nedd4-2, ENaC, and sgk-1 using surface plasmon resonance

Previous studies have characterized interactions between the ubiquitin ligase Nedd4-1 and the epithelial Na⁺ channel (ENaC). Such interactions control the channel cell surface expression and activity. Recently, evidence has been provided that a related protein, termed Nedd4-2, is likely to be the true physiological regulator of the channel. Unlike Nedd4-1, Nedd4-2 also interacts with the aldosterone-induced channel activating kinase sgk-1. The current study uses surface plasmon resonance to quantify the binding of the four WW domains of Nedd4-2 to synthetic peptides corresponding to the PY motifs of ENaC and sgk-1. The measurements demonstrate that WW3 and WW4 are the only Nedd4-2 domains interacting with both ENaC and sgk-1 and that their binding constants are in the 1-6 μM range.     

Analysis of the interaction between human interleukin-5 and the soluble domain of its receptor using a surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor was used to study the interaction of human interleukin-5 (hIL5) with its receptor. IL5 is a major growth factor in the production and activation of eosinophilis. The receptor for IL5 is composed of two subunits, α and β. The α subunit provides the specificity for IL5 and consist of an extracellular soluble domain, a single transmembrane region and a cytoplasmic tail. We expressed the soluble domain of the human IL5 receptor α subunit (shIL5Rα) and human IL5 (hIL5) in Drosophila. Both hIL5 and shIL5Rα were immobilized separately through amine groups onto the carboxylated dextran layer of sensor chips of the BIAcore? (Pharmacia) SPR biosensor after N-hydroxysuccinimide/carbodiimide activation of the chip surface. Interactions were measured for the complementary macromolecule, either shIL5Rα or hIL5, in solution. Kinetics of binding of soluble analyst to immobilized ligand were measured and from this the association rate constant, dissociation rate constant and equilibrium dissociation constant (K_d) were derived. With immobilized shIL5Rα and soluble hIL5, the measured K_d was 2 nM . A similar value was obtained by titration calorimetry. The K_d for Drosophila expressed receptor and IL5 is higher than the values reported for proteins expressed in different systems, likely due to differences in the methods of interaction analysis used for differences in protein glycosylation. Receptor-IL5 binding was relatively pH independent between pH 6.5 and 9.5. Outside this range the dissociation rate increased with compressibility little increased in association rate. The values obtained for the interaction of hIL5 and shIL5Rα were found to depend on which component was immobilized; the K_d was 5.5 nM with immobilized hIL5 and soluble shIL5Rα. The SPR biosensor provides a unified methodology to measure the interaction properties of shIL5Rα and hIL5 derivatives, mutants and mimetic as well as to evaluate potential antagonists of the receptor-cytokine interaction.     

The binding of cytochrome c to neuroglobin: a docking and surface plasmon resonance study

It has recently been proposed that the role of neuroglobin in the protection of neurons from ischaemia induced cell death requires the formation of a transient complex with cytochrome c. No such complex has yet been isolated. Here, we present the results of soft docking calculations, which indicate one major binding site for cytochrome c to neuroglobin. The results yield a plausible structure for the most likely complex structure in which the hemes of each protein are in close contact. NMR analysis identifies the formation of a weak complex in which the heme group of cytochrome c is involved. surface plasmon resonance studies provide a value of 45muM for the equilibrium constant for cytochrome c binding to neuroglobin, which increases significantly as the ionic strength of the solution increases. The temperature dependence of the binding constant indicates that the complex formation is associated with a small unfavourable enthalpy change (1.9kcalmol(-1)) and a moderately large, favourable entropy change (14.8calmol(-1)deg(-1)). The sensitivity of the binding constant to the presence of salt suggests that the complex formation involves electrostatic interactions.     

Identification by surface plasmon resonance of the mycobacterial lipomannan and lipoarabinomannan domains involved in binding to CD14 and LPS-binding protein

The mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), regulate host defence mechanisms through their interaction with pattern recognition receptors such as Toll-like receptors (TLRs). We have developed a surface plasmon resonance assay to analyse the molecular basis for the recognition of Mycobacterium kansasii LM or LAM, by immobilized CD14 and LPS-binding protein (LBP) both being capable to promote presentation of bacterial glycolipids to TLRs. The affinity of either LM/LAM was higher to CD14 than to LBP. Kinetic and Scatchard analyses were consistent with a model involving a single class of binding sites. These interactions required the lipidic anchor, but not the carbohydrate domains, of LM or LAM. We also provide evidence that addition of recombinant LBP enhanced the stimulatory effect of LM or LAM on matrix metalloproteinase-9 expression and secretion in macrophages, through a TLR1/TLR2-dependent mechanism.     

Commensal pathogens, with a focus on Streptococcus pneumoniae, and interactions with the human host

Many important pathogens have humans as their normal ecological niche where healthy carriage dominates over disease. The ability of these commensal pathogens, such as Streptococcus pneumoniae, to cause disease depends on a series of microbial factors as well as of genetic and environmental factors in the human host affecting the clearing capacity mediated by the innate and adaptive immune system. This delicate interplay between microbe and host affects not only the likelihood for a commensal pathogen to cause disease, but also disease type and disease severity.     

Specific binding of integrin alphaIIbbeta3 to RGD peptide immobilized on a nitrilotriacetic acid chip: a surface plasmon resonance study

Nitrilotriacetic acid has been routinely used in protein purification for its high affinity for His-tagged protein in the presence of Ni²⁺. Here we reported a type of nitrilotriacetic acid chip (NTA-chip) prepared by transferring NTA-DOGS containing a lipid monolayer to a 50 nm thick gold layer deposited on a glass slide. The surface binding ability of His-tagged protein and regeneration of NTA chip were characterized using a synthetic polypeptide P1 (His-His-His-His-His-His--aminohexanoic-Gly-Gly-Arg-Gly-Asp-Ser). The effect of divalent cations on integrin binding affinity for RGD ligand was investigated after P1 had been immobilized onto the sensor chip. The results show that the NTA-chip is a useful tool to immobilize His-tagged protein on the chip surface, and can provide a functional orientation for further investigation. The results also show that removing of Ca²⁺ bound on low affinity sites or adding of Mn²⁺ can increase the binding ability of integrin.     

Bottom-up effects may not reach the top: the influence of ant-aphid interactions on the spread of soil disturbances through trophic chains

Soil disturbances that increase nutrient availability may trigger bottom-up cascading effects along trophic chains. However, the strength and sign of these effects may depend on attributes of the interacting species. Here, we studied the effects of nutrient-rich refuse dumps of the leaf-cutting ant, Acromyrmex lobicornis, on the food chain composed of thistles, aphids, tending ants and aphid natural enemies. Using stable isotopes tracers, we show that the nitrogen accumulated in refuse dumps propagates upward through the studied food chain. Thistles growing on refuse dumps had greater biomass and higher aphid density than those growing in adjacent soil. These modifications did not affect the structure of the tending ant assemblage, but were associated with increased ant activity. In contrast to the expectations under the typical bottom-up cascade effect, the increase in aphid abundance did not positively impact on aphid natural enemies. This pattern may be explained by both an increased activity of tending ants, which defend aphids against their natural enemies, and the low capacity of aphid natural enemies to show numerical or functional responses to increased aphid density. Our results illustrate how biotic interactions and the response capacity of top predators could disrupt bottom-up cascades triggered by disturbances that increase resource availability.     

Differential roles of ionic, aliphatic, and aromatic residues in membrane-protein interactions: a surface plasmon resonance study on phospholipases A2

The roles of cationic, aliphatic, and aromatic residues in the membrane association and dissociation of five phospholipases A(2) (PLA(2)), including Asp-49 PLA(2) from the venom of Agkistrodon piscivorus piscivorus, acidic PLA(2) from the venom of Naja naja atra, human group IIa and V PLA(2)s, and the C2 domain of cytosolic PLA(2), were determined by surface plasmon resonance analysis. Cationic interfacial binding residues of A. p. piscivorus PLA(2) (Lys-10) and human group IIa PLA(2) (Arg-7, Lys-10, and Lys-16), which mediate electrostatic interactions with anionic membranes, primarily accelerate the membrane association. In contrast, an aliphatic side chain of the C2 domain of cytosolic PLA(2) (Val-97), which penetrates into the hydrophobic core of the membrane and forms hydrophobic interactions, mainly slows the dissociation of membrane-bound protein. Aromatic residues of human group V PLA(2) (Trp-31) and N. n. atra PLA(2) (Trp-61, Phe-64, and Tyr-110) contribute to both membrane association and dissociation steps, and the relative contribution to these processes depends on the chemical nature and the orientation of the side chains as well as their location on the interfacial binding surface. On the basis of these results, a general model is proposed for the interfacial binding of peripheral proteins, in which electrostatic interactions by ionic and aromatic residues initially bring the protein to the membrane surface and the subsequent membrane penetration and hydrophobic interactions by aliphatic and aromatic residues stabilize the membrane-protein complexes, thereby elongating the membrane residence time of protein.     

Investigations on unconventional hydrogen bonds in RNA binding proteins: the role of CH...O=C interactions

We have investigated the roles played by C-H...O=C interactions in RNA binding proteins. There was an average of 78 CH...O=C interactions per protein and also there was an average of one significant CH...O=C interactions for every 6 residues in the 59 RNA binding proteins studied. Main chain-Main chain (MM) CH...O=C interactions are the predominant type of interactions in RNA binding proteins. The donor atom contribution to CH...O=C interactions was mainly from aliphatic residues. The acceptor atom contribution for MM CH...O=C interactions was mainly from Val, Phe, Leu, Ile, Arg and Ala. The secondary structure preference analysis of CH...O=C interacting residues showed that, Arg, Gln, Glu and Tyr preferred to be in helix, while Ala, Asp, Cys, Gly, Ile, Leu, Lys, Met, Phe, Trp and Val preferred to be in strand conformation. Most of the CH...O=C interacting polar amino acid residues were solvent exposed while, majority of the CH...O=C interacting non polar residues were excluded from the solvent. Long and medium-range CH...O=C interactions are the predominant type of interactions in RNA binding proteins. More than 50% of CH...O=C interacting residues had a higher conservation score. Significant percentage of CH...O=C interacting residues had one or more stabilization centers. Sixty-six percent of the theoretically predicted stabilizing residues were also involved in CH...O=C interactions and hence these residues may also contribute additional stability to RNA binding proteins.     

Studies on the membrane interactions of the cyclotides kalata B1 and kalata B6 on model membrane systems by surface plasmon resonance

In this study we have demonstrated the interactions of kalata B1 and its naturally occurring analogue kalata B6 with five model lipid membranes and have analyzed the binding kinetics using surface plasmon resonance. Two kalata peptides showed a higher affinity for the phosphatidylethanolamine-containing membranes, indicating that the peptides would bind selectively to bacterial membranes. Also we have optimized the procedure for the immobilization of five liposome mixtures and have shown that the procedure provides reproducible levels of immobilized liposomes and could be used to screen the selective binding of putative antimicrobial peptides to model mammalian or microbial phospholipid membranes.     

An analysis of substrate binding interactions in the heme peroxidase enzymes: A structural perspective

The interactions of heme peroxidase enzymes with their substrates have been studied for many years, but only in the last decade or so has structural information begun to appear. This review looks at crystal structures for a number of heme peroxidases in complex with a number of (mainly organic) substrates. It examines the nature and location of the binding interaction, and explores functional similarities and differences across the family.     

Promiscuous binding of ligands by beta-lactoglobulin involves hydrophobic interactions and plasticity

Bovine beta-lactoglobulin (betaLG) binds a variety of hydrophobic ligands, though precisely how is not clear. To understand the structural basis of this promiscuous binding, we studied the interaction of betaLG with palmitic acid (PA) using heteronuclear NMR spectroscopy. The titration was monitored using tryptophan fluorescence and a HSQC spectrum confirmed a 1:1 stoichiometry for the PA-betaLG complex. Upon the binding of PA, signal disappearances and large changes in chemical shifts were observed for the residues located at the entrance and bottom of the cavity, respectively. This observation indicates that the lower region makes a rigid connection with PA whereas the entrance is more flexible. The result is in contrast to the binding of PA to intestinal fatty acid-binding protein, another member of the calycin superfamily, in which structural consolidation occurs upon ligand binding. On the other hand, the ability of betaLG to accommodate various hydrophobic ligands resembles that of GroEL, in which a large hydrophobic cavity and flexible binding site confer the ability to bind various hydrophobic substrates. Considering these observations, it is suggested that, in addition to the presence of the hydrophobic cavity, the plasticity of the entrance region makes possible the binding of hydrophobic ligands of various shapes. Thus, in contrast to the specific binding seen for many enzymes, betaLG provides an example of binding with low specificity but high affinity, which may play an important role in protein-ligand and protein-protein networks.     

Alpha-1-proteinase inhibitor is a heparin binding serpin: molecular interactions with the Lys rich cluster of helix-F domain

Alpha-1-proteinase (alpha-1-PI) inhibitor is the major circulating serine protease inhibitor in humans. The porcine elastase and trypsin inhibitory activity of human and ovine alpha-1-PI is activated several fold in the presence of anti-coagulant heparin. The activation is allosteric and appears to be characterized by two steps of binding; a weak followed by a strong binding. The Kass for ovine and human alpha-1-PI inhibition of porcine pancreatic elastase was increased approximately 45 fold and 38 fold respectively. Using a combinatorial approach of multiple sequence alignment, surface topology, chemical modification and tryptic peptide mapping to identify the sequence of the heparin bound peptide; we demonstrate that heparin binds to the lysyl rich region of the F-helix of alpha-1-PI, which differs from that of heparin-antithrombin (AT) interactions. Molecular docking prediction using the MEDock algorithm approximates the three positively charged lysines (K154, K155, K174) of human alpha-1-PI in this interaction. This heparin alpha-1-PI interaction has been exploited to develop an affinity purification method, which can be used universally to obtain homogenous preparations of mammalian alpha-1-PIs useful for augmentation therapy. Collectively, all these findings imply that alpha-1-PI has a major role in regulating extra cellular protease activity and the physiological activator is heparin.