An immuno-chemo-proteomics method for drug target deconvolution

Chemical proteomics is an emerging technique for drug target deconvolution and profiling the toxicity of known drugs. With the use of this technique, the specificity of a small molecule inhibitor toward its potential targets can be characterized and information thus obtained can be used in optimizing lead compounds. Most commonly, small molecules are immobilized on solid supports and used as affinity chromatography resins to bind targets. However, it is difficult to evaluate the effect of immobilization on the affinity of the compounds to their targets. Here, we describe the development and application of a soluble probe where a small molecule was coupled with a peptide epitope which was used to affinity isolate binding proteins from cell lysate. The soluble probe allowed direct verification that the compound after coupling with peptide epitope retained its binding characteristics. The PKC-alpha inhibitor Bisindolylmaleimide-III was coupled with a peptide containing the FLAG epitope. Following incubation with cellular lysates, the compound and associated proteins were affinity isolated using anti-FLAG antibody beads. Using this approach, we identified the known Bisindolylmaleimide-III targets, PKC-alpha, GSK3-beta, CaMKII, adenosine kinase, CDK2, and quinine reductase type 2, as well as previously unidentified targets PKAC-alpha, prohibitin, VDAC and heme binding proteins. This method was directly compared to the solid-phase method (small molecule was immobilized to a solid support) providing an orthogonal strategy to aid in target deconvolution and help to eliminate false positives originating from nonspecific binding of the proteins to the matrix.     

Analysis of the soluble ATP-binding proteome of plant mitochondria identifies new proteins and nucleotide triphosphate interactions within the matrix

The interactions of ATP inside plant mitochondria were investigated by identifying the soluble nucleotide binding proteome captured using immobilized ATP. Selected proteins were separated by 1D SDS-PAGE and 2D IEF-SDS-PAGE and identified by ESI-Q-TOF MS/MS. A range of highly enriched proteins were identified from the mitochondrial proteome, including 14-3-3 proteins and RNA binding proteins, as well as proteins known to contain nucleotide binding domains and/or to be inhibited or stimulated by ATP.     

IgG binding kinetics to oligo B protein A domains on lipid layers immobilized on a 27 MHz quartz-crystal microbalance

Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x = 1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27 MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (Delta m(max)), association constants (K(a)), association and dissociation rate constants (k(1) and k(-1), respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains.     

Chemical proteomic study of isoprenoid chain interactome with a synthetic photoaffinity probe

A chemical proteomic approach was developed for profiling the noncovalent interactome of isoprenoid chain in the yeast proteome. A chemical probe that harbors a biotin moiety and a photoreactive benzophenone group linked to the terminal of geranyl group was synthesized. Photoaffinity labeling was performed by incubating the Saccharomyces cerevisiae proteome and the probe under 365 nm UV light. Thirty proteins were identified by immobilized NeutraAvidin enrichment, on-bead digestion, online 2-D nano-LC/MS/MS identification and semi-quantitative proteomic analysis. As noted by Gene Ontology annotation, the identified proteins demonstrate a wide range of catalytic activity in several biological processes, especially in metabolism and biosynthesis. Further data analysis shows that hydrophobic binding of the synthetic probe is potentially the major interaction force leading to covalent labeling. These results argue that intracellular allosteric interactions conferred by the isoprenoid chain of the corresponding chemical structures may be widespread at an interactomic level.     

Engineered protein a for the orientational control of immobilized proteins

This work describes the genetic engineering and characterization of a histidine-tagged fragment of protein A. The histidine tag results in the site-selective immobilization of the protein A receptor and the preservation of its high ligand affinity when immobilized on solid supports. The fragment was expressed at high yield in E. coli and purified to homogeneity. When selectively immobilized to histidine binding matrices, the protein A fragment exhibits high affinity for soluble IgG. We further demonstrate from adsorption isotherms that the receptor exhibits a homogeneous, high affinity population at densities where steric crowding between large ligands does not affect the apparent receptor affinity. This engineered receptor is appropriate for a range of applications including sensor design or those using immobilized Fc-tagged proteins.     

Binding and signaling of surface-immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins

Development of biosensor devices typically requires incorporation of the molecular recognition element into a solid surface for interfacing with a signal detector. One approach is to immobilize the signal transducing protein directly on a solid surface. Here we compare the effects of two direct immobilization methods on ligand binding, kinetics, and signal transduction of reagentless fluorescent biosensors based on engineered periplasmic binding proteins. We used thermostable ribose and glucose binding proteins cloned from Thermoanaerobacter tengcongensis and Thermotoga maritima, respectively. To test the behavior of these proteins in semispecifically oriented layers, we covalently modified lysine residues with biotin or sulfhydryl functions, and attached the conjugates to plastic surfaces derivatized with streptavidin or maleimide, respectively. The immobilized proteins retained ligand binding and signal transduction but with adversely affected affinities and signal amplitudes for the thiolated, but not the biotinylated, proteins. We also immobilized these proteins in a more specifically oriented layer to maleimide-derivatized plates using a His(2)Cys(2) zinc finger domain fused at either their N or C termini. Proteins immobilized this way either retained, or displayed enhanced, ligand affinity and signal amplitude. In all cases tested ligand binding by immobilized proteins is reversible, as demonstrated by several iterations of ligand loading and elution. The kinetics of ligand exchange with the immobilized proteins are on the order of seconds.     

Specific phospholipid recognition by human immunodeficiency virus type-1 neutralizing anti-gp41 2F5 antibody

HIV-1 neutralizing monoclonal antibody (Mab) 2F5 recognizes a membrane-partitioning gp41 sequence. Just recently its capacity to react with cardiolipin has been demonstrated. Here, we have studied the specificity of Mab2F5-phospholipid interactions comparing partitioning into lipid bilayers with recognition of molecular species dispersed in solution. Using a liposome-based ELISA we demonstrate a preferential association with cardiolipin bilayers. When different soluble lysoderivatives were compared in their capacity to inhibit Mab2F5 binding to immobilized HIV-1 peptide epitope, only dilysocardiolipin resulted effective in blocking the process. Dilyso-cardiolipin also competed with native-functional gp41 for 2F5 recognition. Thus, our data support specific cardiolipin recognition by 2F5 that is not dependent on lipid bilayer assembly and involves the epitope-binding site. These findings might be of relevance for understanding the molecular basis of HIV-1 immune evasion.     

Binding kinetics of soluble ligands to transmembrane proteins: comparing an optical biosensor and dynamic flow cytometry

BACKGROUND: The kinetics of protein-protein interactions can be monitored with optical biosensors based on the principles of either surface plasmon resonance or mirror resonance. These methods are straightforward for soluble proteins, but not for proteins inserted in the plasma membrane. METHODS: We monitored with an IASys biosensor system, based on a resonant mirror: (1) the binding of cells to an immobilized ligand, (2) the binding of a soluble ligand to immobilized cells, and (3) the binding of a soluble ligand to immobilized plasma membrane vesicles. For comparison, the kinetics of fluorescent antibody binding to intact cells were measured by dynamic flow cytometry. RESULTS: With an optical biosensor, the useful configuration is the one based on immobilized plasma membrane vesicles. However, signals can be detected only for very abundant binding sites (>10(6) per cell). Dynamic flow cytometry allows the accurate determination of the k(on) and k(off) of antibody binding. The sensitivity of the method is two orders of magnitude better than with an optical biosensor. CONCLUSIONS: Although biosensors constitute a method of choice for measuring the interactions between soluble proteins, they are not well suited for measuring the interaction between soluble proteins and membrane-embedded proteins. On the contrary, flow cytometry is well suited for such an application, when it is used in a dynamic mode.     

Nuclear export of the yeast mRNA-binding protein Nab2 is linked to a direct interaction with Gfd1 and to Gle1 function

Nuclear export of mRNA is mediated by interactions between soluble factors and nuclear pore complex (NPC) proteins. In Saccharomyces cerevisiae, Nab2 is an essential RNA-binding protein that shuttles between the nucleus and cytoplasm. The mechanism for trafficking of Nab2-bound mRNA through the NPC has not been defined. Gle1 is also required for mRNA export, and Gle1 interactions with NPC proteins, the RNA helicase Dbp5, and Gfd1 have been reported. Here we report that Nab2, Gfd1, and Gle1 associate in a complex. By using immobilized recombinant Gfd1, Nab2 was isolated from total yeast lysate. A similar biochemical assay with immobilized recombinant Nab2 resulted in coisolation of Gfd1 and Gle1. A Nab2-Gfd1 complex was also identified by coimmunoprecipitation from yeast lysates. In vitro binding assays with recombinant proteins revealed a direct association between Nab2 and Gfd1, and two-hybrid assays delineated Gfd1 binding to the N-terminal Nab2 domain. This N-terminal Nab2 domain is distinct from its RNA binding domains suggesting Nab2 could bind Gfd1 and RNA simultaneously. As Nab2 export was blocked in a gle1 mutant at the restrictive temperature, we propose a model wherein Gfd1 serves as a bridging factor between Gle1 and Nab2-bound mRNA during export.     

Design of a specific peptide tag that affords covalent and site-specific enzyme immobilization catalyzed by microbial transglutaminase

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with beta-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (k(cat)) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.     

Identification of Possible Lipid Binding Regions in Food Proteins and Peptides and Additional <Emphasis Type="Italic">In Silico</Emphasis> Analysis

The results of the search for potential helical lipid binding regions in a number of well-known food proteins is described. All selected food proteins have either well-described or strong indications of protein-lipid interaction features. The results are obtained with the aid of a number of selected bioinformatics tools. The identified potential lipid binding regions either correspond nicely with regions demonstrated experimentally or can be identified as novel lipid binding regions. The results are discussed in relation to earlier found experimental results and if relevant are discussed in mechanistic terms as well. A possible general use of the approach as used in this study is discussed as well. The in this study described approach can be used as a tool to come to a more focused approach for further research of yet unknown proteins. It is also possible to use this approach for more directed research when it comes to for example rational functional design of proteins for particular desired features like the emulsifying or foaming ability of the protein of interest.     

Expression of multivalency in the affinity chromatography of antibodies. Appendix: Derivation and evaluation of equations for independent bivalent interacting systems in quantitative affinity chromatography.

The expression of multivalency in the interaction of antibody with immobilized antigen was evaluated by quantitative affinity chromatography. Zones of radioisotopically labeled bivalent immunoglobulin A monomer derived from the myeloma protein TEPC 15 were eluted from columns of phosphorylcholine-Sepharose both in the absence and presence of competing soluble phosphorylcholine. At sufficient immobilized phosphorylcholine concentration, the variation of elution volume of bivalent monomer with soluble ligand was found to deviate from that observed for the univalent binding of the corresponding Fab fragment. In addition, the apparent binding affinity of the bivalent monomer increased with immobilized antigen density. Use of equations relating the variation of elution volume with free ligand concentration for a bivalent binding protein allowed calculation of microscopic single-site binding parameters for the bivalent monomeric antibody to both immobilized and soluble phosphorylcholine. The chromatographic data not only demonstrate the effect of multivalency on apparent binding affinity but also offer a relatively simple means to measure microscopic dissociation constants for proteins participating in bivalent interactions with their ligands.     

Binding, reconstitution and 2D crystallization of membrane or soluble proteins onto functionalized lipid layer observed in situ by reflected light microscopy

Monolayer of functionalized lipid spread at the air/water interface is used for the structural analysis of soluble and membrane proteins by electron crystallography and single particle analysis. This powerful approach lacks of a method for the screening of the binding of proteins to the surface of the lipid layer. Here, we described an optical method based on the use of reflected light microscopy to image, without the use of any labeling, the lipid layer at the surface of buffers in the Teflon wells used for 2D crystallization. Images revealed that the lipid layer was made of a monolayer coexisting with liposomes or aggregates of lipids floating at the surface. Protein binding led to an increase of the contrast and the decrease of the fluidity of the lipid surface, as demonstrated with the binding of soluble Shiga toxin B subunit, of purple membrane and of solubilized His-BmrA, a bacterial ABC transporter. Moreover the reconstitution of membrane proteins bound to the lipidic surface upon detergent removal can be followed through the appearance of large recognizable domains at the surface. Proteins binding and reconstitution were further confirmed by electron microcopy. Overall, this method provides a quick evaluation of the monolayer trials, a significant reduction in screening by transmission electron microscopy and new insights in the proteins binding and 2D crystallogenesis at the lipid surface.     

Use of dextrans as long and hydrophilic spacer arms to improve the performance of immobilized proteins acting on macromolecules

New dextran-agarose supports, suitable for covalent immobilization of enzymes and proteins acting on macromolecular substrates, were prepared. The thick internal fibers of agarose gels were covered by a low-density layer of long, flexible, hydrophilic, and inert dextran molecules. Rennin and protein A were immobilized on these novel supports and the resulting derivatives exhibited a very high capacity for biological recognition of soluble macromolecular substrates. Caseinolytic activity of this immobilized enzyme was 15-fold higher than activity of directly immobilized rennin, through short spacer arms, on agarose gels. Similarly, the new derivatives of immobilized protein A were able to adsorb up to 2 molecules of immunoglobulin per each molecule of immobilized protein A. When the immobilized proteins were secluded away from the support surface by using these new long and hydrophilic spacer arms, they exhibit minimal steric hindrances that could be promoted by the proximity of the support surface.     

Hydrophobicity of transmembrane proteins: spatially profiling the distribution

A hallmark of soluble globular protein tertiary structure is a hydrophobic core and a protein exterior populated predominantly by hydrophilic residues. Recent hydrophobic moment profiling of the spatial distribution of 30 globular proteins of diverse size and structure had revealed features of this distribution that were comparable. Analogous profiling of the hydrophobicity distribution of the alpha-helical buried bundles of several transmembrane proteins, as the lipid/protein interface is approached from within the bilayer, reveals spatial hydrophobicity profiles that contrast with those obtained for the soluble proteins. The calculations, which enable relative changes of hydrophobicity to be simply identified over the entire spatial extent of the multimer within the lipid bilayer, show the accumulated zero-order moments of the bundles to be mainly inverted with respect to that found for the soluble proteins. This indicates a statistical increase in the average residue hydrophobic content as the lipid bilayer is approached. This result differs from that of a relatively recent calculation and qualitatively agrees with earlier calculations involving lipid exposed and buried residues of the alpha-helices of transmembrane proteins. Spatial profiling, over the entire spatial extent of the multimer with scaled values of residue hydrophobicity, provides information that is not available from calculations using lipid exposure alone.     

A novel microtiter plate based method for identification of B‐cell epitopes

A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B‐cell epitopes in two different proteins using a peptide‐based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15‐mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor‐α, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non‐covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor‐α were added to identify potential linear B‐cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non‐covalent binding although the peptides were in fact present on the non‐covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B‐cell epitopes in protein antigens. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Immobilization of alpha(1)-acid glycoprotein for chromatographic studies of drug-protein binding

A new method for preparing immobilized alpha1-acid glycoprotein (AGP) for use in drug-protein binding studies was developed and optimized. In this approach, periodate was used under mild conditions to oxidize the carbohydrate chains in AGP for attachment to a hydrazide-activated support. The final conditions chosen for this oxidation involved the reaction of 5.0 mg/mL AGP at 4 degrees C and pH 7.0 with 5-20 mM periodic acid for 10 min. These conditions helped maximize the immobilization of AGP without significantly affecting its activity. This method was evaluated by using it to attach AGP to silica for use in high-performance affinity chromatography and self-competition zonal elution studies. In work with R- and S-propranolol, only one type of binding site was observed for both enantiomers on the immobilized AGP, in agreement with previous studies using soluble AGP. The association equilibrium constants measured for the immobilized AGP with R- and S-propranolol at pH 7.4 and 37 degrees C were 2.7 x 10(6) and 4.2 x 10(6) M(-1), respectively, with linear van't Hoff plots being obtained between 5 and 37 degrees C. Work performed with other drugs also gave good agreement between the behavior seen for immobilized AGP and that for soluble AGP. The same immobilization method described in this work could be used to attach AGP to other materials, such as those used for surface plasmon resonance or alternative biosensors.     

Proteomic profiling of hempseed proteins from Cheungsam

Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp (Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3-10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development.     

Surface plasmon resonance imaging analysis of hexahistidine-tagged protein on the gold thin film coated with a calix crown derivative

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the hexahistidine-ubiquitin-tagged human parathyroid hormone fragment (His₆-Ub-hPTHF(1–34)) expressed inEscherichia coli. The hexahistidine-specific antibody was immobilized on a thin gold film coated with ProLinker^TM B, a novel calixcrown derivative with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrices. The soluble and insoluble fractions of anE. coli cell lysate were spotted onto the antibody-coated gold chip, which was then washed with buffer (pH 7.4) solution and dried. SPR imaging measurements were carried out to detect the expressed His₆-Ub-hPTHF (1–34). There was no discernible protein image in the uninduced cell lysate, indicating that non-specific binding of contaminant proteins did not occur on the gold chip surface. It is expected that the approach used here to detect affinity-tagged recombinant proteins using an SPR imaging technique could be used as a powerful tool for the analyses of a number of proteins in a high-throughput mode.     

Investigation of the 'switch-epitope' concept with random peptide libraries displayed as thioredoxin loop fusions.

The 'FLITRX' random peptide library, consisting of dodecamer loop peptides displayed on a thioredoxin-flagellin scaffold on Escherichia coli, was used to select peptide sequences with affinity for a monoclonal antibody. These peptides were further screened for pH- and metal-sensitive antibody binding. Several zinc-sensitive peptides were identified, termed 'switch epitopes'. A soluble, monomeric thioredoxin loop ('Trxloop') insertion analog of a FLITRX switch epitope was constructed and its antibody binding properties were characterized by Western blots. Zinc-dependent antibody recognition was maintained in the Trxloop protein although the apparent antibody affinity was lower. This Trxloop protein bound to an immobilized metal affinity chromatography matrix, similar to a 'histidine-patch' thioredoxin variant, and was reversibly precipitated by 1 mM Zn(2+) or Cu(2+) ions. Residues important for zinc and antibody binding were determined by site-directed mutagenesis. The Trxloop antibody affinity was increased by saturation mutagenesis. Biotinylated Trxloop ('Biotrxloop') variants of the original and improved affinity Trxloop proteins were constructed and characterized by surface plasmon resonance measurements. Increased antibody affinity was partially due to a slower antibody desorption rate, although the relative adsorption rates were dependent on the amount of immobilized Biotrxloop protein, indicating an influence of avidity on the apparent affinity.