Tresyl activation of a hydroxyalkyl ligand for coupling to a hydrazide gel: stable immobilization of T-2 toxin for affinity purification of T-2 antibody

A stable T-2 hydrazide gel is prepared by activating T-2 toxin with tresyl chloride followed by coupling to agarose-adipic acid hydrazide. Utilized as an affinity chromatography column, this T-2 hydrazide gel purifies a monoclonal antibody for T-2 in high yield directly from ascites fluid. Specific antibody trapped on the column is eluted either with excess T-2 or at pH 11.6. Much less successful are two other T-2 affinity columns that were prepared and evaluated: T-2 bovine serum albumin Affi-Gel 15 and T-2 hexylamine Sepharose.     

Activation of Sepharose with epichlorohydrin and subsequent immobilization of ligand for affinity adsorbent.

The optimal conditions for the activation of Sepharose by epichlorohydrin and subsequent immobilization of ligands were investigated. Under the optimal conditions for activation, namely, 30% Sepharose-5% epichlorohydrin-0.4 M NaOH, 40 degrees C, 2 h, the maximum amount of epoxy group was introduced into Sepharose with low cross-linking. The absorbents obtained by using N-acetyl-D-glucosamine, tri-N-acetylchitotriose, and glycoprotein as a ligand exhibited no nonspecific adsorption and good permeability for the high molecular substance to be purified, and were stable in an alkaline solution. Solanum tuberosum agglutinin was specifically adsorbed on a tri-N-acetylchitotriose-Sepharose column and was quantitatively recovered by elution with 0.2 M ammonia solution. Furthermore, the column could be repeatedly used under these conditions without reduction of its capacity.     

Manipulation of ligand binding affinity by exploitation of conformational coupling

Traditional approaches for increasing the affinity of a protein for its ligand focus on constructing improved surface complementarity in the complex by altering the protein binding site to better fit the ligand. Here we present a novel strategy that leaves the binding site intact, while residues that allosterically affect binding are mutated. This method takes advantage of conformationally distinct states, each with different ligand-binding affinities, and manipulates the equilibria between these conformations. We demonstrate this approach in the Escherichia coli maltose binding protein by introducing mutations, located at some distance from the ligand binding pocket, that sterically affect the equilibrium between an open, apo-state and a closed, ligand-bound state. A family of 20 variants was generated with affinities ranging from an approximately 100-fold improvement (7.4 nM) to an approximately two-fold weakening (1.8 mM) relative to the wild type protein (800 nM).     

Discovery of a dopamine D4 selective PET ligand candidate taking advantage of a click chemistry based REM linker

Employing D4 selective azaindoles as lead compounds, a focused library of the carbocyclic arene bioisosteres 1 was synthesized when we took advantage of the click chemistry derived triazolylmethyl acrylate resin 2. Ligand binding assays on monoaminergic GPCRs led to SARs that indicated further lead structure optimizations when the attachment of alkoxy substituents provided both an improvement of the biological properties and the opportunity to introduce (18)F as a radioisotope. Finally, radiosynthesis resulted in formation of the radioligand [(18)F]7h that showed optimal logD(7.4) of 2.8 and was determined to be highly stable in human serum. Thus, [(18)F]7h represents a promising dopamine D4 selective radioligand for positron emission tomography (PET).     

Macroporous polyacrylamide monolithic gels with immobilized metal affinity ligands: the effect of porous structure and ligand coupling chemistry on protein binding

Macroporous polyacrylamide gels (MPAAG) with iminodiacetic acid (IDA) functionality were prepared by (i) chemical modification of polyacrylamide gel, (ii) co-polymerization of acrylamide with allyl glycidyl ether (AGE) and N,N'metylene-bis(acrylamide) (MBAAm) followed by coupling IDA ligand or (iii) by copolymerization of acrylamide and MBAAm with functional monomer carrying IDA-functionality (1-(N,N-bis(carboxymethyl)amino-3-allylglycerol). Screening for optimized conditions for the production of the MPAAG with required porous properties was performed in a 96-well chromatographic format that allowed parallel production and analysis of the MPAAG prepared from reaction mixtures with different compositions. Scanning electron microscopy of the fabricated MPAAG revealed two different types of the porous structures: monomodal macroporous structure with large interconnected pores separated by dense non-porous pore walls in case of plain gels or gels produced via copolymerization with AGE. The other type of the MPAAG (gel produced via co-polymerization with functional monomer carrying IDA-functionality) had bimodal pore structure with large interconnected pores separated by the pore walls pierced through with micropores. The effect of different modifications of MPAAG monoliths and of porous structure of the MPAAG (monomodal and bimodal porous structure) on protein binding has been evaluated.     

Mutation in a flexible linker modulates binding affinity for modular complexes

Tandem beta zippers are modular complexes formed between repeated linear motifs and tandemly arrayed domains of partner proteins in which β-strands form upon binding. Studies of such complexes, formed by LIM domain proteins and linear motifs in their intrinsically disordered partners, revealed spacer regions between the linear motifs that are relatively flexible but may affect the overall orientation of the binding modules. We demonstrate that mutation of a solvent exposed side chain in the spacer region of an LHX4–ISL2 complex has no significant effect on the structure of the complex, but decreases binding affinity, apparently by increasing flexibility of the linker.     

Functionalization of oligosaccharide mimetics and multimerization using squaric diester-mediated coupling

Functionalized carbohydrate-centered glycoclusters formed the starting material for the synthesis of tagged oligosaccharide and glycoconjugate mimetics, which were obtained by thiourea-bridging, peptide coupling and in particular squaric diester-mediated coupling. The latter method could also be utilized to provide new multivalent glycoconjugates, which were tested for their anti-adhesive properties in an ELISA with Escherichia coli bacteria.     

Rational engineering of a fluorescein-binding anticalin for improved ligand affinity

The anticalin FluA is an artificial lipocalin with novelspecificity for the fluorescein group, which was engineered from an insect bilin-binding protein by targeted random mutagenesis and selection. Based on the crystal structure of FluA, an attempt was made to improve the complementarity of its ligand pocket to fluorescein by rational protein design. Several side chains participating in sub-optimal interactions with the ligand were identified and replaced by residues that promised a better steric fit. As a result, the substitution of Ala45 by Ile and of Ser114 by Thr or Arg led to a tight affinity of ca. 1 nM, which is approximately 30-fold better than that of the parental anticalin. Similar to the original FluA, the improved version shows almost complete quenching of the bound ligand fluorescence. Interestingly, the quenching effect was significantly reduced when Trp129 was replaced by Tyr, thus supporting the previously postulated role of this residue, which closely packs against the bound ligand, for efficient electron transfer to the excited fluorescein. Circular dichroism spectra revealed that all variants investigated had retained the lipocalin fold. Corresponding thermal unfolding experiments confirmed similar folding stabilities, with melting temperatures ranging from 52.9 to 60.5 degrees C (i.e., for the high-affinity variant).     

A novel concept for ligand attachment to oligonucleotides via a 2'-succinyl linker

Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects. In this report, a new method for synthesizing oligonucleotide conjugates is described. 2′-Amino-2′-deoxy-5′-dimethoxytrityl-uridine was select ively acylated with a succinic acid linker at the 2′ position. This compound was incorporated at the 3′ end of an oligonucleotide corresponding to the sequence of Oblimersen. The carboxyl group was protected for oligonucleotide synthesis as a benzyl ester, which could be selectively cleaved at the solid phase by a catalytic phase transfer reaction using palladium nanoparticles as catalyst. An oligonucleotide–fluorescein conjugate was prepared by condensation of aminofluorescein. Circular dichroism spectroscopic experiments showed a B-DNA type structure. The melting temperature of the duplex was only slightly lower than that of Oblimersen. Biological activity measured by western blotting resulted in a Bcl-2 target downregulation nearly identical to that of control Oblimersen on human melanoma cells, proving that this method is attractive for the binding of ligands located in the minor groove.     

The effect of linker length on binding affinity of a photoswitchable molecular glue for DNA

Molecular glue for DNA is a small synthetic ligand that adheres two single-stranded DNAs to produce a double-stranded DNA. We previously devised a photoswitchable molecular glue (PMG) that uses external light stimuli to reversibly control DNA hybridization. To optimize the structure of PMG, we synthesized a series of PMGs and evaluated the effect of changing the methylene linker length on the binding affinity and photoresponse. From the comprehensive T_m and CSI-TOF-MS measurements, a PMG possessing a three-methylene linker with carbamate linkage produced maximum binding affinity and photoswitching ability. These results indicate that a small difference in the linker can significantly affect PMG function. These findings are useful for designing new photoswitchable DNA-binding ligands.     

Prediction of protein interdomain linker regions by a hidden Markov model

MOTIVATION: Our aim was to predict protein interdomain linker regions using sequence alone, without requiring known homology. Identifying linker regions will delineate domain boundaries, and can be used to computationally dissect proteins into domains prior to clustering them into families. We developed a hidden Markov model of linker/non-linker sequence regions using a linker index derived from amino acid propensity. We employed an efficient Bayesian estimation of the model using Markov Chain Monte Carlo, Gibbs sampling in particular, to simulate parameters from the posteriors. Our model recognizes sequence data to be continuous rather than categorical, and generates a probabilistic output. RESULTS: We applied our method to a dataset of protein sequences in which domains and interdomain linkers had been delineated using the Pfam-A database. The prediction results are superior to a simpler method that also uses linker index.     

The derivatization of oxidized polysaccharides for protein immobilization and affinity chromotography

The present report describes the preparation of modified polysaccharides matrices useful for the synthesis of affinity adsorbents and immobilized proteins. Hydrazido-matrices were synthesized by condensing an excess of the bifunctional reagent, adipic acid dihydrazide, with periodate oxidized cellulose paper, Sephadex, or Sepharose matrices. Ribonucleotide dialdehyde cofactors, glyceraldehyde 3-phosphate, pyridoxal 5′-phosphate and oxidized DNAase B were separately bound to the hydrazido-polymers. Azido-matrices obtained by modification of the hydrazido-derivatives were coupled to specific amino ligands such as amino acids and proteins. Several adsorbents were prepared and used as models for affinity chromatography.     

Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker

The sequence-specific affinity chromatographic isolation of plasmid DNA from crude lysates of E. coli DH5alpha fermentations is addressed. A zinc finger-GST fusion protein that binds a synthetic oligonucleotide cassette containing the appropriate DNA recognition sequence is described. This cassette was inserted into the SmaI site of pUC19 to enable the affinity isolation of the plasmid. It is shown that zinc finger-GST fusion proteins can bind both their DNA recognition sequence and a glutathione-derivatized solid support simultaneously. Furthermore, a simple procedure for the isolation of such plasmids from clarified cell lysates is demonstrated. Cell lysates were clarified by cross-flow Dean vortex microfiltration, and the permeate was incubated with zinc finger-GST fusion protein. The resulting complex was adsorbed directly onto glutathione-Sepharose. Analysis of the glutathione-eluted complex showed that plasmid DNA had been recovered, largely free from contamination by genomic DNA or bacterial cell proteins.     

Poly(vinylpyrrolidone) for bioconjugation and surface ligand immobilization

Poly(vinylpyrrolidone) (PVP), a nonionic and nontoxic polymer with antifouling properties, has been synthesized via RAFT polymerization to obtain thiol-terminated PVP. We demonstrate that when the polymer is adsorbed onto the surface of colloidal silica particles, the terminal thiol groups of PVP remain accessible for chemical modification and lend themselves to the immobilization of ligands. We show that ligand attachment onto the surface via conjugation to PVP is reversible, as the polymer can be desorbed from the surface for conjugate and surface recovery. We present the conjugation of a model peptide and an oligonucleotide to PVP via the polymer terminal thiol and demonstrate that conjugates remain functional in molecular recognition assay. The developed technique offers a novel method to functionalize low-fouling surfaces for a variety of biomedical applications and presents opportunities to use PVP as a macromolecular drug carrier.     

Synthesis and estrogen receptor affinity of a 4-hydroxytamoxifen-labeled ligand for diagnostic imaging

A 10-step synthesis of a novel 4-hydroxytamoxifen-DTPA ligand (HOTam-DTPA) is reported. Tamoxifen and its primary metabolite 4-hydroxytamoxifen are common estrogen receptor ligands. Consequently, tamoxifen has found utility as the targeting component of various diagnostic agents for selective imaging of estrogen receptor-rich tissue, specifically breast cancer. An L-aspartic acid-derived DTPA analogue was attached to the ethyl side chain of 4-hydroxy-tamoxifen using N,N'-dimethylethylenediamine as a hydrophilic linker. A competitve estrogen receptor binding assay using [3H]-17beta-estradiol was performed to determine the effect of the ethyl side chain modification on estrogen receptor affinity. The results show that while the relative affinity of HOTam-DTPA for the estrogen receptor is approximately 10-fold lower than that of tamoxifen, it still remains a potent ligand at relatively low concentrations.     

Avidin column as a highly efficient and stable alternative for immobilization of ligands for affinity chromatography

The avidin/biotin system was applied as a general mediator in the adsorption/desorption or immobilization of biologically active macromolecules to solid supports. In this context, model biotinylated proteins (lectins and antibodies) were attached to avidin-coupled Sepharose. As examples for affinity chromatography, peanut agglutinin and anti-transferrin antibody were used to isolate asialofetuin and transferrin, respectively. The capacity and product yields were significantly better than those achieved with conventional affinity chromatography on CNBr-activated Sepharose columns containing the same lectin or antibody. Moreover, the columns were characterized by improved stability properties exhibiting remarkably low levels of leakage.     

Dual immobilization of biomolecule on the glass surface using cysteine as a bifunctional linker

A novel method for highly efficient enzyme immobilization on the glass surface, by incorporating cysteine as a linker has been demonstrated. The internal glass surface of test tube was pretreated with (3-mercaptopropyl) trimethoxysilane sol–gel and cysteine capped silver nanoparticles, to generate a cysteine layer. This, cysteine rich surface is then used to covalently immobilize alkaline phosphatase on both groups (amino and carboxyl) of cysteine through carbodiimide and glutaraldehyde treatment. The cysteine capped silver nanoparticles were synthesized with an average nanoparticle size of 61 nm as determined by particle size analyzer, while cysteine capping of nanoparticles was confirmed by Fourier transform infra-red spectroscopy. Enhanced enzymatic activity of about 73% was obtained using the dual immobilization technique, while 40% enzyme activity was recovered with carboxyl group and 51% with amino group only. The re-usability of the enzyme immobilized test tube was found to be 8 times and the enzyme retained 85% of its initial activity. With such high immobilization efficiency, cysteine provides a new approach for enhanced immobilization and its integration into different industrial processes and biosensor technology.     

Light-induced activation of an inert surface for covalent immobilization of a protein ligand.

A simple and mild procedure is developed for the preparation of an activated polymer surface, used for immobilization of a protein ligand through a covalent linkage. Activation of the polymer surface is carried out by attaching an active functional group through 1-fluoro-2-nitro-4-azidobenzene (FNAB). UV irradiation of FNAB transforms its azido group into a highly reactive nitrene, which binds with the inert polymer surface, whereas the active fluoro group of FNAB, now part of the polymer, remains intact. Covalent linkage between the ligand and the inert surface is established through this active fluoro group in a thermochemical reaction. The photochemical step is carried out under dry conditions to exclude the possibility of undesirable reactions between the solvent and the highly reactive nitrene. The method can be used for activation of different inert polymer surfaces having carbon hydrogen bonds. The efficacy of our method is demonstrated by immobilizing horseradish peroxidase on an activated polystyrene surface. The enzyme, immobilized through the photolinker, is found to give a twofold increase in absorbance with the substrate as compared to the directly adsorbed enzyme. The method may have many applications in the preparation of bioreactors, biostrips, and biosensors, and in diagnostic tests involving the ELISA technique.     

Distinct affinity and effector residues in the binding site for a regulatory ligand

A hypothesis concerning two distinct classes of amino acid residues in some regulatory binding sites is proposed. The affinity residues are those that are unable to transduce the ligand information signal but are responsible for overcoming the barrier for the attachment of a ligand to its binding site while the effector residues transfer the binding signal to the other functional part of the protein, which then undergoes a non-equilibrium energetic cycle induced by interaction with the ligand.As an example, the purine nucleotide inhibition of H⁺ transport through the uncoupling protein of brown adipose tissue mitochondria is discussed; there is a concentration range in which the nucleotide is bound but does not inhibit H⁺ transport. This is interpreted in terms of inaccessibility of the effector residues inducing H⁺ transport inhibition below a certain threshold concentration.