Investigating the binding behaviour of two avidin‐based testosterone binders using molecular recognition force spectroscopy

Molecular recognition force spectroscopy, a biosensing atomic force microscopy technique allows to characterise the dissociation of ligand–receptor complexes at the molecular level. Here, we used molecular recognition force spectroscopy to study the binding capability of recently developed testosterone binders. The two avidin‐based proteins called sbAvd‐1 and sbAvd‐2 are expected to bind both testosterone and biotin but differ in their binding behaviour towards these ligands. To explore the ligand binding and dissociation energy landscape of these proteins, we tethered biotin or testosterone to the atomic force microscopy probe while the testosterone‐binding protein was immobilized on the surface. Repeated formation and rupture of the ligand–receptor complex at different pulling velocities allowed determination of the loading rate dependence of the complex‐rupturing force. In this way, we obtained the molecular dissociation rate (k_off) and energy landscape distances (x_β) of the four possible complexes: sbAvd‐1‐biotin, sbAvd‐1‐testosterone, sbAvd‐2‐biotin and sbAvd‐2‐testosterone. It was found that the kinetic off‐rates for both proteins and both ligands are similar. In contrast, the x_β values, as well as the probability of complex formations, varied considerably. In addition, competitive binding experiments with biotin and testosterone in solution differ significantly for the two testosterone‐binding proteins, implying a decreased cross‐reactivity of sbAvd‐2. Unravelling the binding behaviour of the investigated testosterone‐binding proteins is expected to improve their usability for possible sensing applications. Copyright © 2014 John Wiley & Sons, Ltd.     

Reversible surface thiol immobilization of carboxyl group containing haptens to a BIAcore biosensor chip enabling repeated usage of a single sensor surface

We describe a reversible immobilization method for carboxyl group containing haptens that makes the repeated usage of a BIAcore biosensor chip possible. Haptens which are immobilized according to the surface thiol method can be removed completely from the sensor surface again by a reducing step. In the first part of our study, analogues of the herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid were immobilized in succession to a biosensor surface of a BIAcore surface plasmon resonance instrument according to the thiol coupling method. Direct kinetic analysis of these ligands to a polyclonal anti-2,4-dichlorophenoxyacetic acid antibody were performed using these biosensor surfaces. In the second part of the study, different amounts of 2,4-dichlorophenoxyacetic acid were sequentially immobilized onto the same biosensor surface in order to generate a calibration plot for 2,4-dichlorophenoxyacetic acid. Using this plot, the quantitative detection of the herbicide down to a concentration of 0.1 microg/mL, the maximum admissible concentration of pesticides in drinking water, is possible.     

Love–Hate ligands for high resolution analysis of strain in ultra-stable protein/small molecule interaction

The pathway of ligand dissociation and how binding sites respond to force are not well understood for any macromolecule. Force effects on biological receptors have been studied through simulation or force spectroscopy, but not by high resolution structural experiments. To investigate this challenge, we took advantage of the extreme stability of the streptavidin–biotin interaction, a paradigm for understanding non-covalent binding as well as a ubiquitous research tool. We synthesized a series of biotin-conjugates having an unchanged strong-binding biotin moiety, along with pincer-like arms designed to clash with the protein surface: ‘Love–Hate ligands’. The Love–Hate ligands contained various 2,6-di-ortho aryl groups, installed using Suzuki coupling as the last synthetic step, making the steric repulsion highly modular. We determined binding affinity, as well as solving 1.1–1.6 Å resolution crystal structures of streptavidin bound to Love–Hate ligands. Striking distortion of streptavidin’s binding contacts was found for these complexes. Hydrogen bonds to biotin’s ureido and thiophene rings were preserved for all the ligands, but biotin’s valeryl tail was distorted from the classic conformation. Streptavidin’s L3/4 loop, normally forming multiple energetically-important hydrogen bonds to biotin, was forced away by clashes with Love–Hate ligands, but Ser45 from L3/4 could adapt to hydrogen-bond to a different part of the ligand. This approach of preparing conflicted ligands represents a direct way to visualize strained biological interactions and test protein plasticity.     

The influence of transport on the kinetics of binding to surface receptors: application to cells and BIAcore

Accurate estimation of biomolecular reaction rates from binding data, when ligands in solution bind to receptors on the surfaces of cells or biosensors, requires an understanding of the contributions of both molecular transport and reaction. Efficient estimation of parameters requires relatively simple models. In this review, we give conditions under which various transport effects are negligible and identify simple binding models that incorporate the effects of transport, when transport cannot be neglected. We consider effects of diffusion of ligands to cell or biosensor surfaces, flow in a BIAcore biosensor, and distribution of receptors in a dextran layer above the sensor surface. We also give conditions under which soluble receptors can be expected to compete effectively with surface-bound receptors.     

A polymerase chain reaction-based ribosomal DNA detection technique using a surface plasmon resonance detector for a red tide causing microalga, Alexandrium affine

A detection technique with a DNA probe was developed for the bloom‐forming alga Alexandrium affine harvested in Japan. The design of this probe was based on the sequence polymorphism within the 28S ribosomal DNA (rDNA) of this strain using the BIAcore? 2000 biosensor, which determines surface plasmon resonance. The specific DNA sequence in 28S rDNA for A. affine was determined by sequence data analysis, and a probe was designed for the detection of A. affine. A fragment of the 28S rDNA from A. affine was amplified by polymerase chain reaction and applied to the BIAcore? sensor system, and the target DNA was selectively recognized by species‐specific hybridization using two DNA probes: a fluorescein isothiocyanate (FITC)‐labeled probe and a biotin‐labeled DNA probe. Using FITC‐labeled anti‐immunogloblin G antibody, enhancement of the response for the target DNA can be detected directly as a resonant unit change. In this detection method, a difference within only 20 base pairs of the target could be detected, and specific detection of A. affine was achieved intraspecifically.     

Synthesis of biotinylated glycoconjugates and their use in a novel ELISA for direct comparison of HIV-1 Gp120 recognition of GalCer and related carbohydrate analogues

As part of our program directed toward the design and synthesis of high-affinity ligands for the GalCer-binding site on the HIV cell surface glycoprotein, gp120, we required a reliable method for qualitatively assessing relative binding affinities for related analogues. Due to the hydrophilic nature of these synthetic conjugates, difficulties were encountered with typical ELISA methods, which rely upon hydrophobic interactions to anchor the ligand to a microtiter plate. Other types of assays were also problematic due to nonspecific binding of gp120. Therefore, we developed a general method for plating water-soluble ligands on microtiter plates using biotin/NeutrAvidin recognition for adhesion. A water-soluble GalCer analogue was prepared by conjugating psychosine to biotin using a novel tetraethylene glycol linker. In a similar manner, LacCer and GlcCer analogues were prepared and these conjugates were plated into microtiter wells containing NeutrAvidin. Unoccupied sites were blocked using biotin functionalized as a primary amide. Gp120 binding to galactosyl sphingosine, GalSph (19), GlcSph (22), and LacSph (23) conjugates was assessed through incubation with recombinant HRP-gp120. It was determined that LacSph has the strongest interaction with gp120. The binding affinities of GalSph and GlcSph were similar to each other and less strong than LacSph. These data contradict earlier studies where HPTLC showed that LacCer and GlcCer do not significantly bind gp120. They also contradict liposome-based assays that reported psychosine is not recognized by gp120. The extent of plating for each biotinylated molecule was quantified using HRP-biotin, allowing direct comparison of ligand plating efficiencies for the first time. Several other synthetic biotin conjugates were prepared and tested, demonstrating the feasibility of performing ELISA on water-soluble ligands.     

Effect of biolinker on the detection of prostate specific antigen in an interferometry

Prostate-specific antigen (PSA) has been identified as a significant biomarker for prostate cancer screening. Heavily-doped porous silicon, etched to form a Fabry-Perot fringe pattern, can be applied to an interferometric sensing for detecting PSA bound with PSA-antibody. In the previous works, a calyx crown derivative (Prolinker-A) was used as an alternative biolinker on the porous silicon surface for interferometric biosensing of DNA-damaging chemical instead of employing the conventional biomolecular affinity method using biotin, which resulted in a denser linker formation. In this study, G5 amineterminated PAMAM (poly amidoamine) dendrimer as a biolinker, was applied to an interferometric sensing of PSA by using porous silicon, which shows the enhancement of adhesion capability and increase of functional groups more than those with Prolinker-A. Considerably low level down to 1 ng/mL of PSA could be detected by this sensing system.     

Identification of tubular heparan sulfate as a docking platform for the alternative complement component properdin in proteinuric renal disease

Properdin binds to proximal tubular epithelial cells (PTEC) and activates the complement system via the alternative pathway in vitro. Cellular ligands for properdin in the kidney have not yet been identified. Because properdin interacts with solid-phase heparin, we investigated whether heparan sulfate proteoglycans (HSPG) could be the physiological ligands of properdin. Kidneys from proteinuric rats showed colocalization of syndecan-1, a major epithelial HSPG, and properdin in the apical membranes of PTEC, which was not seen in control renal tissue. In vitro, PTEC did not constitutively express properdin. However, exogenous properdin binds to these cells in a dose-dependent fashion. Properdin binding was prevented by heparitinase pretreatment of the cells and was dose-dependently inhibited by exogenous heparin. ELISA and surface plasmon resonance spectroscopy (BIAcore) showed a strong dose-dependent interaction between heparan sulfate (HS) and properdin (K(d) = 128 nm). Pretreatment of HSPG with heparitinase abolished this interaction in ELISA. Competition assays, using a library of HS-like polysaccharides, showed that sulfation pattern, chain length, and backbone composition determine the interaction of properdin with glycosaminoglycans. Interestingly, two nonanticoagulant heparin derivatives inhibited properdin-HS interaction in ELISA and BIAcore. Incubation of PTEC with human serum as complement source led to complement activation and deposition of C3 on the cells. This C3 deposition is dependent on the binding of properdin to HS as shown by heparitinase pretreatment of the cells. Our data identify tubular HS as a novel docking platform for alternative pathway activation via properdin, which might play a role in proteinuric renal damage. Our study also suggests nonanticoagulant heparinoids may provide renoprotection in complement-dependent renal diseases.     

Antibody-antigen interactions measured by surface plasmon resonance: global fitting of numerical integration algorithms

The interactions between adenylate kinase (AK) and a monoclonal antibody against AK (McAb3D3) were examined by means of optical biosensor technology, and the sensograms were fitted to four models using numerical integration algorithms. The interaction of a solution of McAb3D3 with immobilized AK follows a double exponential function and the data fitted well to an inhomogeneous ligand model. The interaction of a solution AK with immobilized McAb3D3 follows a single exponential function and the data fitted well to a pseudo-first order reaction model. The true association constants of AK binding to McAb3D3 in solution were obtained from competition BIAcore measurements. The difference in results obtained with solid-phase BIAcore and competition BIAcore may be due to rebinding of the dissociated analyte to the immobilized surface. The results obtained with BIAcore are compared to those obtained by ELISA methods. We suggest that the best method for analysis of BIAcore data is direct, global fitting of sensorgrams to numerical integration algorithms corresponding to the different possible models for binding.     

Characterization and application of a surface modification designed for QCM-D studies of biotinylated biomolecules

The rapid development of surface sensitive biosensor technologies, especially towards nanoscale devices, requires increasing control of surface chemistry to provide reliable and reproducible results, but also to take full advantage of the sensing opportunities. Here, we present a surface modification strategy to allow biotinylated biomolecules to be immobilized to gold coated sensor crystals for quartz crystal microbalance with dissipation monitoring (QCM-D) sensing. The unique feature of QCM-D is its sensitivity to nanomechanical (viscoelastic) properties at the sensing interface. The surface modification was based on mixed monolayers of oligo(ethylene glycol) (OEG) disulfides, with terminal -OH or biotin groups, on gold. Mixtures containing 1% of the biotin disulfide were concluded to be the most appropriate based on the performance when streptavidin was immobilized to biotinylated sensors and the subsequent biotinylated bovine serum albumin (BSA) interaction was studied. The OEG background kept the unspecific protein binding to a minimum, even when subjected to serum solutions with a high protein concentration. Based on characterization by contact angle goniometry, ellipsometry, and infrared spectroscopy, the monolayers were shown to be well-ordered, with the OEG chains predominantly adopting a helical conformation but also partly an amorphous structure. Storage stability was concluded to depend mainly on light exposure while almost all streptavidin binding activity was retained when storing the sensors cold and dark for 8 weeks. The surface modification was also tested for repeated antibody-antigen interactions between BSA and anti-BSA (immobilized to biotinylated protein A) in QCM-D measurements lasting for >10h with intermediate basic regeneration. This proved an excellent stability of the coating and good reproducibility was obtained for 5 interaction cycles. With this kind of generic surface modification QCM-D can be used in a variety of biosensing applications to provide not only mass but also relevant information of the structural properties of adlayers.     

A surface plasmon resonance immunosensor based on the streptavidin-biotin complex.

It is shown that a streptavidin monolayer immobilized onto an evaporated gold film with biotin forms the basis of a highly specific sensing element. As an example, we show that by immobilizing the biotinylated antibody sex hormone binding globulin (alpha-SHBG) to the bound streptavidin monolayer a specific sensor for the antigen SHBG is readily fabricated. The interaction between immobilized antibody and corresponding antigen is monitored by surface plasmon resonance spectroscopy and is shown to follow a classic Langmuir isotherm. Detection of SHBG at nanomolar concentrations is demonstrated.     

Application of peptide nucleic acid to the direct detection of deoxyribonucleic acid amplified by polymerase chain reaction.

Double-stranded DNA amplified by polymerase chain reaction (PCR) was detected by peptide nucleic acid (PNA) using a BIAcore 2000 biosensor based on surface plasmon resonance (SPR). PNA is an artificial oligo amide that is capable of forming highly stable complexes with complementary oligonucleotides. We succeeded in the direct detection of double-stranded DNA, amplified by PCR with high-sequence specificity. It was shown that the target DNA was available for detection over the range of 40-160 nM. Therefore, the detection limit was 7.5 pmol of the target DNA (143 bases, applied volume 30 microliters). Our DNA detection system, the combination of BIAcore and the probe PNA, could detect the target DNA with good reproducibility. In this report, we show that our system is a powerful tool for the diagnosis of pathologically significant DNA.     

Ligand-modified aminobisphosphonate for linking proteins to hydroxyapatite and bone surface

An increase in bone resorption is one of the main symptoms of osteoporosis, a disease that affects more and more individuals every day. Bisphosphonates are known to inhibit bone resorption and thus are being used as a treatment for osteoporosis. Aminobisphosphonates present a functionality that can be easily used for conjugation to other molecules, such as peptides, proteins, and ligands for protein recognition. In this study, an aminobisphosphonate conjugated with biotin was used as a model linker for protein attachment to bone. With this system, the interaction of biotinylated aminobisphosphonate with hydroxyapatite, a major mineral component of bone, was investigated. Quantification of the binding of aminobisphosphonate to hydroxyapatite was performed using a fluorescently labeled antibody for biotin. Additionally, the interaction of the biotinylated aminobisphosphonate with multiple treatments of cortical bone from the midshaft of a cow femur was studied. It was demonstrated that modified aminobisphosphonate reagents can bind hydroxyapatite and bone at high levels, while the biotin functionality is free to be recognized by the fluorescently labeled antibiotin antibody, suggesting that modified aminobisphosphonates could be used to link other peptides or proteins to the bone surface.     

Methods for site controlled coupling to carboxymethyldextran surfaces in surface plasmon resonance sensors

In the present study, optimized methods for in-situ ligand immobilization to carboxymethylated dextran coated gold surfaces for use in surface plasmon resonance (SPR) based biosensor devices were developed. Immobilization methods based on selective thiol reactions, either via thiols on the sensor surface or via thiols introduced on the ligand, were shown to give high yields of functionally active material. Methods are also described for linking aldehyde containing ligands to hydrazide modified surfaces and for coupling based on the avidin/biotin concept. The importance of blocking residual active groups (for thiol coupling) and reduction of acid labile hydrazone bonds formed in the aldehyde coupling was demonstrated. The methods were found to work efficiently in combination with conditions suitable for electrostatic preconcentration of ligands to be coupled, a concept we earlier developed for coupling of amine containing ligands (Löfås & Johnsson, 1990).     

Design,production, and characterization of a monomeric streptavidin and its application for affinity purification of biotinylated proteins

To expand the application of the streptavidin-biotin technology for reversible affinity purification of biotinylated proteins, a novel form of monomeric streptavidin was engineered and produced using Bacillus subtilis as the expression host. By changing as little as two amino acid residues (T90 and D128) to alanine, the resulting mutant streptavidin designated DM3 was produced 100% in the monomeric form as a soluble functional protein via secretion. It remained in the monomeric state in the presence or absence of biotin. Interaction of purified monomeric streptavidin with biotin was studied by surface plasmon resonance-based BIAcore biosensor. Its on-rate is comparable to that of monomeric avidin while its off-rate is seven times lower. The dissociation constant was determined to be 1.3 x 10(-8)M. These properties make it an attractive agent for affinity purification of biotinylated proteins. An affinity matrix with immobilized DM3 mutein was prepared and applied to purify biotinylated cytochrome c from a crude extract. Biotinylated cytochrome c could be purified to homogeneity in one step and was shown to retain full biological activity. Advantages of using DM3 mutein over other traditional methods in the purification of biotinylated proteins are discussed.     

Microfluidic protein detection through genetically engineered bacterial cells

Protein microarray technology, in which a large number of capture ligands are spatially arrayed at a high density, presents an attractive method for high-throughput proteomic analysis. Toward this end, we demonstrate the first cell-based protein detection in a microsystem, wherein Escherichia coli cells are genetically engineered to express the desired capture proteins on the membrane surface and are spatially arrayed as sensing elements in a microfluidic device. An E. coli clone expressing peptide ligands with high affinity and high specificity for target molecules was isolated a priori. Then these cells were electrokinetically immobilized on gold electrodes using dielectrophoresis, thus allowing each sensor element to be electrically addressable. Flow cytometry and subsequent fluorescence analysis verified the highly specific capture and detection of target molecules by the bacteria. Finally, through the coexpression of peptide-based capture ligands on the cell surface and fluorescent protein in the cytoplasm, we demonstrate an effective means of directly linking the fluorescence intensity to the density of capture ligands.     

Transport effects on surface–volume biological reactions

Many cellular reactions involve a reactant in solution binding to or dissociating from a reactant confined to a surface. This is true as well for a BIAcore, an optical biosensor that is widely used to study the interaction of biomolecules. In the flow cell of this instrument, one of the reactants is immobilized on a flat sensor surface while the other reactant flows past the surface. Both diffusion and convection play important roles in bringing the reactants into contact. Usually BIAcore binding data are analyzed using well known expressions that are valid only in the reaction-limited case when the Damk?hler number Da is small. Asymptotic and singular perturbation techniques are used to analyze dissociation of the bound state when Da is small and O(1). Linear and nonlinear integral equations result from the analysis; explicit and asymptotic solutions are constructed for physically realizable cases. In addition, effective rate constants are derived that illustrate the effects of transport on the measured rate constants. All these expressions provide a direct way to estimate the rate constants from BIAcore binding data.     

A plasmid expression system for quantitative in vivo biotinylation of thioredoxin fusion proteins in Escherichia coli.

The high affinity binding interaction of biotin to avidin or streptavidin has been used widely in biochemistry and molecular biology, often in sensitive protein detection or protein capture applications. However, in vitro chemical techniques for protein biotinylation are not always successful, with some common problems being a lack of reaction specificity, inactivation of amino acid residues critical for protein function and low levels of biotin incorporation. This report describes an improved expression system for the highly specific and quantitative in vivo biotinylation of fusion proteins. A short 'biotinylation peptide', described previously by Schatz, is linked to the N-terminus of Escherichia coli thioredoxin (TrxA) to form a new protein, called BIOTRX. The 'biotinylation peptide' serves as an in vivo substrate mimic for E. coli biotin holoenzyme synthetase (BirA), an enzyme which usually performs highly selective biotinylation of E.coli biotin carboxyl carrier protein (BCCP). A plasmid expression vector carrying the BIOTRX and birA genes arranged as a bacterial operon can be used to obtain high level production of soluble BIOTRX and BirA proteins and, under appropriate culture conditions, BIOTRX protein produced by this system is completely biotinylated. Fusions of BIOTRX to other proteins or peptides, whether these polypeptides are linked to the C-terminus or inserted into the BIOTRX active site loop, are also quantitatively biotinylated. Both types of BIOTRX fusion can be captured efficiently on avidin/streptavidin media for purification purposes or to facilitate interaction assays. We illustrate the utility of the system by measurements of antibody and soluble receptor protein binding to BIOTRX fusions immobilized on streptavidin-conjugated BIAcore chips.     

Structure-function studies of Escherichia coli biotin synthase via a chemical modification and site-directed mutagenesis approach.

In Escherichia coli, biotin synthase (bioB gene product) catalyzes the key step in the biotin biosynthetic pathway, converting dethiobiotin (DTB) to biotin. Previous studies have demonstrated that BioB is a homodimer and that each monomer contains an iron-sulfur cluster. The purified BioB protein, however, does not catalyze the formation of biotin in a conventional fashion. The sulfur atom in the iron-sulfur cluster or from the cysteine residues in BioB have been suggested to act as the sulfur donor to form the biotin molecule, and yet unidentified factors were also proposed to be required to regenerate the active enzyme. In order to understand the catalytic mechanism of BioB, we employed an approach involving chemical modification and site-directed mutagenesis. The properties of the modified and mutated BioB species were examined, including DTB binding capability, biotin converting activity, and Fe(2+) content. From our studies, four cysteine residues (Cys 53, 57, 60, and 97) were assigned as the ligands of the iron-sulfur cluster, and Cys to Ala mutations completely abolished biotin formation activity. Two other cysteine residues (Cys 128 and 188) were found to be involved mainly in DTB binding. The tryptophan and histidine residues were suggested to be involved in DTB binding and dimer formation, respectively. The present study also reveals that the iron-sulfur cluster with its ligands are the key components in the formation of the DTB binding site. Based on the current results, a refined model for the reaction mechanism of biotin synthase is proposed.